Vaccinia virus H3L and B5R specific monoclonal antibodies and methods of making and using same
The invention relates to antibodies and subsequences thereof that specifically bind to poxvirus B5R envelope protein, antibodies and subsequences thereof that specifically bind to pox virus H3L envelope protein, and combinations thereof.
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This application claims priority to U.S. Ser. No. 61/127,729, filed May 14, 2008, U.S. Ser. No. 60/979,028, filed Oct. 10, 2007 and PCT/US08/78316, filed Sep. 30, 2008, which are expressly incorporated herein by reference.
TECHNICAL FIELDThe invention relates to antibodies and subsequences thereof that specifically bind to poxvirus B5R envelope protein, antibodies and subsequences thereof that specifically bind to poxvirus H3L envelope protein, and combinations thereof. The invention also relates to methods of providing a subject with protection against poxvirus infection or pathogenesis including infectious and pathogenic poxviruses (e.g., variola major and variola minor smallpox, monkeypox, cowpox, vaccinia, molluscum contagiosum and camelpox) using antibodies or subsequences thereof that specifically bind to poxvirus B5R envelope protein, antibodies or subsequences thereof that specifically bind to poxvirus H3L envelope protein, and combinations of antibodies or subsequences thereof that specifically bind to poxvirus B5R envelope protein with antibodies or subsequences thereof that specifically bind to poxvirus H3L envelope protein. Furthermore, the invention relates to methods of protecting or decreasing susceptibility of a subject to a poxvirus infection or pathogenesis including infectious and pathogenic poxviruses (e.g., variola major and variola minor smallpox, monkeypox, cowpox, molluscum contagiosum and camelpox) and small pox vaccine viruses using antibodies or subsequences thereof that specifically bind to poxvirus B5R envelope protein, antibodies or subsequences thereof that specifically bind to poxvirus H3L envelope protein, and combinations of antibodies or subsequences thereof that specifically bind to poxvirus B5R envelope protein with antibodies or subsequences thereof that specifically bind to poxvirus H3L envelope protein.
IntroductionSmallpox is a highly lethal viral infection of humans (30% mortality) (Fenner et al., World Health Organization, Geneva; Henderson et al., JAMA 281:2127 (1999)), which can spread rapidly through a population. Smallpox is a top bioterrorism concern, and is frequently generally considered the #1 bioterrorism danger (Henderson et al., JAMA 281:2127 (1999); LeDuc and Jahrling, Emerg. Infect. Dis. 7:155 (2001); Meltzer et al., Emerg. Infect. Dis. 7:959 (2001); O'Toole et al., Clin. Infect. Dis. 34:972 (2002)). The smallpox vaccine consists of live vaccinia virus and is the gold standard of vaccines since it has led to the complete eradication of wild smallpox from the human population. Renewed fears that smallpox might be deliberately released in an act of bioterrorism have led to a resurgence in the study of treatment of smallpox (variola virus) infection, rare but severe side effects of the smallpox vaccine (vaccinia virus, VACV), and treatment of other poxviruses such as monkeypox. Individuals under the age of 35 (approximately 50% of the population) have not been vaccinated against smallpox, leaving them highly susceptible in the event of an outbreak. Furthermore, there is an active smallpox vaccination campaign in the USA military, and VIG (Vaccinia Immune Globulin,) is used to treat the rare side effects of vaccination. Finally, in 2003, a monkeypox outbreak occurred for the first time in the USA (Huhn et al., Clin. Infect. Dis. 41:1742 (2005)).
Currently, VIG (Vaccinia Immune Globulin) is the only licensed therapeutic to treat the side effects of smallpox vaccination (DryVax immunization), and it is the treatment available in case of an actual smallpox or monkeypox outbreak/bioterrorism event (Hopkins and Lane, Clin. Infect. Dis. 39:819 (2004); Wittek, R., Int. J. Infect. Dis. 10:193 (2006)). Unfortunately, VIG is a poorly characterized, highly variable, human product that is only available in very limited quantities and is of limited potency (Hopkins and Lane, Clin. Infect. Dis. 39:819 (2004); Wittek, R., Int. J. Infect. Dis. 10:193 (2006)). Each of these issues is a major problem for biodefense preparedness against a smallpox bioterrorism event. Problems with VIG—particularly the small number of VIG doses available and their limited potency—have led to great interest in the development of a better anti-smallpox immunotherapy.
Poxviruses (vaccinia, variola/smallpox, monkeypox) have two virions forms, Intracellular Enveloped Virions (IMV) and Extracellular Enveloped Virions (EEV), each with distinct biology and numerous different surface proteins (Condit et al., Adv. Virus Res. 66:31 (2006); Smith et al., J. Gen. Virol. 83:2915 (2002)). As such, an understanding of the virion structures is required to develop knowledge regarding the targets of protective antibodies.
The most abundant viral particle (up to 99% of total) is the intracellular mature virion (IMV), which accumulates in infected cells and is released as cells die (Moss, B., Poxyiridae: The Viruses and their Replication, In Fundamental Virology, D. M. Knipe, and P. Howley, eds. (2001)). IMVs are environmentally stable infectious virus particles and likely represent the principle virion type involved in transmission between hosts. An alternate morphogenesis pathway is taken by a proportion of IMVs inside infected cells. These immature virions become wrapped in a double membrane from the trans-Golgi and are then translocated to the cell surface where the outermost membrane fuses with the plasma membrane (Roos et al., EMBO J. 15:2343 (1996)). These virions may be released from the cell surface as extracellular enveloped virus (EEV) (Smith et al., J. Gen. Viral. 83:2915 (2002)). EEV are more fragile and less abundant than the IMV, and are considered to be primarily involved in dissemination within the same host rather than transmission between hosts (Lustig et al., J. Virol. 79:13454 (2005); Payne, L. G, J. Gen. Virol. 50:89 (1980); Smith et al., J. Gen. Viral. 83:2915 (2002))
In humans, high neutralizing antibody titers have been associated with protective immunity against smallpox infection (Mack, J. E., Pediatr. Nurs. 14:220 (1988); Sarkar et al., Bull World Health Organ 52:307 (1975)). Long-term antibody titers and T cell memory to the smallpox vaccine do not correlate in humans (Crotty et al., J. Immunol. 171:4969 (2003); Hammarlund et al., Nat. Med. 11:1005 (2005)) excluding the possibility that antibody titers were simply a biomarker for memory T cells. Vaccinia Immune Globulin (VIG) is an effective treatment against smallpox, as it was able to reduce the number of smallpox cases ˜80% among exposed individuals in four case controlled studies (Hobday, T. L., Lancet 1:907 (1962); Kempe, C. H., Pediatrics 25:176 (1960); Kempe et al., Pediatrics 18:177 (1956); Marennikova, S. S, Bull World Health Organ 27:325 (1962)).
Vaccinia immune globulin (VIG) is licensed to treat complications following smallpox vaccination and would likely be used in the event of a smallpox or monkeypox outbreak. VIG is produced by purifying and pooling IgG from smallpox vaccine recipients. VIG is tested and treated for blood borne pathogens, and tested for efficacy by in vitro neutralization of VACV and in vivo treatment of SCID mice infected with VACV (CangeneCorporation (2005), Vaccinia Immune Globulin Intravenous (Human) (VIGIV) package insert. F. CBER, ed.; DynportVaccineCompany (2005), Vaccinia Immune Globulin Intravenous (Human) (VIGIV) package insert. F. CBER, ed; Goldsmith et al., Vox Sang 86:125 (2004); Wittek, R., Int. J. Infect. Dis. 10:193 (2006). Recruitment of sufficient donors for production of VIG is a substantial problem, resulting in a small supply of VIG available. Furthermore, the potency of VIG is limited, due to its dilute and polyclonal nature. In addition, there are always safety concerns regarding human blood products. Altogether, these problems with VIG—particularly the small number of VIG doses available and their limited potency—have led to great interest in the development of a better anti-smallpox immunotherapy. These are serious problems with VIG can be solved by production of a high quality mAb product.
SUMMARYHumanAntibodies that bind to poxvirus proteins, such as B5R and H3L, or anti-B5R and anti-H3L monoclonal antibodies and subsequences thereof, are described. Antibodies provided. Exemplary human antibodies were produced by immunizing trans-chromosomic mice (KM Mice™) with soluble recombinant vaccinia virus B5R or H3L protein. Isolated B5R specific antibodies recognize different epitopes on B5R, for example at least two “epitopes” on B5R, as determined by antibody cross-blocking studies. Isolated H3L specific antibodies recognize one or more epitopes on H3L as determined by antibody cross-blocking studies. Antibodies that specifically bind B5R or H3L protein, and combinations of such antibodies, inhibit virus infection in vitro and/or protect mice (BALB/c and SCID) from vaccinia virus (VACVWR and VACVNYCBOH) challenge in in vivo animal models. These results confirm the functional characteristics of human anti-vaccinia virus B5R or H3L monoclonal antibodies and their usefulness alone and in combination as a VIG (vaccinia immune globulin) replacement to treat (e.g., provide a subject with protection against or decrease susceptibility of a subject) poxvirus (e.g., smallpox) infection or as a treatment of adverse side effects or complications associated with or caused by vaccinia virus or poxvirus vaccination, immunization or infection.
The invention therefore provides antibodies, including human, humanized and chimeric B5R or H3L binding antibodies, and compositions including antibodies, such as human, humanized and chimeric B5R or H3L binding antibodies such as pharmaceutical compositions, and kits containing antibody. The invention also provides methods for prophylactic and therapeutic treatment of poxvirus infection and pathogenesis; methods for providing a subject with protection against a poxvirus infection or pathogenesis; and methods for protecting or decreasing susceptibility of a subject to a poxvirus infection and pathogenesis. In various aspects, the poxvirus is a variola major or variola minor smallpox virus. In more particular aspects, the poxvirus is monkeypox, cowpox, vaccinia, molluscum contagiosum or camelpox.
Compositions include fully human, humanized and chimeric (e.g., human/mouse chimera) monoclonal antibodies that recognize and bind to B5R or H3L, or homologs thereof, such as vaccinia B5R, B6 or H3L proteins, or B5R, B6 or H3L protein homolog of a related poxvirus, e.g., such as variola or other poxvirus (e.g., monkeypox). Methods include passive immunization with human, humanized and chimeric (e.g., human/mouse chimera) polyclonal and monoclonal antibodies that bind to B5R or H3L, before or after contact with, exposure to or infection with a poxvirus, or pathogenesis caused by or associated with poxvirus contact, exposure or infection. Methods include treatment methods prior to or before contact with, exposure to or infection with a poxvirus (prophylaxis) as well as treatment methods following contact with, exposure to or infection with a poxvirus (therapeutic) including development of one or more symptoms associated with or caused by poxvirus infection or pathogenesis. Non-limiting examples of symptoms of poxvirus infection or pathogenesis include high fever, fatigue, headache, backache, malaise, rash (maculopapular, vesicular or pustular) or lesions, delirium, vomiting, diarrhea and excess bleeding. Methods of the invention therefore include reducing, decreasing, inhibiting, ameliorating, delaying or preventing onset, progression, severity, duration, frequency, susceptibility or probability of one or more symptoms associated with poxvirus contact, exposure, infection or pathogenesis.
Antibodies that bind to B5R, or H3L, or B5R, or H3L protein homologs are useful for treating a subject having or at risk of having a poxvirus, before infection (prophylaxis) or following infection (therapeutic). The invention therefore provides methods of using antibodies that bind to B5R or H3L in treatment (e.g., therapeutic or prophylactic) of poxvirus infection or pathogenesis.
The invention further provides methods for providing a subject with protection against, or protecting a subject from, poxvirus infection or pathogenesis. In one embodiment, a method includes administering an amount of an antibody that binds to B5R or H3L sufficient to provide the subject with protection against, or protect the subject from, poxvirus infection or pathogenesis.
The invention also provides methods for protecting or decreasing susceptibility of a subject to a poxvirus infection or pathogenesis. In one embodiment, a method includes administering a composition comprising an amount of an antibody that binds to B5R or H3L sufficient to protect or decrease susceptibility of the subject to poxvirus infection or pathogenesis.
The invention additionally provides methods for decreasing or preventing an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus. In one embodiment, a method includes administering a composition comprising a sufficient amount of an antibody that binds B5R or H3L to decrease or prevent an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus. In another embodiment, a method includes administering a composition comprising a sufficient amount of an antibody that binds B5R or H3L to an immune-suppressed or HIV-positive subject to decrease or prevent an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus. In various aspects, adverse side effects or complications decreased or prevented include postvaccinial encephalitis, progressive vaccinia, eczema vaccinatum, generalized vaccinia, accidental infection of close contacts, rashes and periocular infection. In further aspects, the subject is a candidate for or has been vaccinated with a vaccinia virus (e.g., modified vaccinia Ankara (MVA), vaccinia virus Lister strain, vaccinia virus LC16m8 strain, vaccinia virus NYCBOH strain, vaccinia virus Wyeth strain, vaccinia ACAM2000, or vaccinia virus prepared from calf lymph, Dryvax®) or immuinized against a poxvirus. In additional various aspects, the subject is administered the antibody that binds B5R or H3L prior to, concurrently with, following or within 1-2, 2-4, 4-12, 12-24, 24-48, 48-72 hours or, 4, 5, 6, 7, or more days of vaccination with vaccinia virus or immunization against a poxvirus.
Antibodies of the invention can bind to B5R or H3L, optionally present on one or more poxvirus (e.g., infectious or pathogenic poxvirus or live or attenuated vaccinia virus or a related poxvirus such as monkeypox) strains or isolates or species. Thus, the antibodies have one or more effects on virus infectivity, replication, proliferation, titer, or onset, progression, severity, frequency, duration or probability of one or more symptoms, adverse side effects or complications associated with or caused by virus infection or pathogenesis, or vaccination with a vaccinia virus, vaccinia virus protein, or immunization against a poxvirus.
Methods of the invention include methods in which partial or complete protection against poxvirus infection or pathogenesis, or a symptom of poxvirus infection or pathogenesis is provided. In one embodiment, a human, humanized or chimeric B5R or H3L binding antibody inhibits poxvirus infection of a cell in vitro or in vivo, or inhibits poxvirus binding to a cell in vitro or in vivo. In another embodiment, a human, humanized or chimeric B5R or H3L antibody reduces or decreases virus titer, infectivity, replication, proliferation, or an amount of a viral protein of one or more vaccinia virus or poxvirus strains, isolates or species. In yet another embodiment, a human, humanized or chimeric B5R or H3L binding antibody inhibits, delays, or prevents increases in virus titer, infectivity, replication, proliferation, or an amount of a viral protein of one or more vaccinia virus or poxvirus strains, isolates or species. In still another embodiment, a human, humanized or chimeric B5R or H3L binding antibody provides a subject with protection against a poxvirus infection or pathogenesis or protects or decreases susceptibility of a subject to infection or pathogenesis, by one more poxvirus strains, isolates or species. In a further embodiment, a human, humanized or chimeric B5R or H3L binding antibody decreases onset, progression, severity, frequency, duration or probability of one or more symptoms or complications associated with infection or pathogenesis by one or more poxvirus strains or isolates or subtypes. Exemplary symptoms include, for example, high fever, fatigue, headache, backache, malaise, rash (maculopapular, vesicular or pustular) or lesions, delirium, vomiting, diarrhea, and excess bleeding.
B5R or H3L binding antibody can be administered or delivered in accordance with the invention by any suitable in vitro, ex vivo or in vivo method. In various embodiments, a composition is administered prior to, concurrently with, or following contact with or exposure to a poxvirus, poxvirus infection or pathogenesis, or vaccination with a vaccinia virus or immunization against a poxvirus. In various aspects, human, humanized or chimeric B5R or H3L binding antibody is administered or in vivo delivered systemically (e.g., intravenous injection, subcutaneous injection, intravenous infusion, intramuscular injection), regionally, or locally to a subject.
Antibodies of the invention include polyclonal and monoclonal antibodies and mixtures thereof, which can be any of IgG, IgA, IgM, IgE, IgD, and any isotype thereof, for example, IgG1, IgG2, IgG3 or IgG4. Antibodies include intact human, humanized and chimeric immunoglobulin molecules with two full-length heavy chains and two full-length light chains (e.g., mature portion of heavy and light chain variable region sequences) as well as subsequences/fragments of heavy or light chain which retain at least a part of a function of a reference or parental intact antibody that specifically binds B5R or H3L protein or B5R or H3L homolog. Antibody subsequences can have the same or substantially the same binding specificity, binding affinity or anti-poxvirus activity as a reference or parental intact anti-B5R or H3L binding antibody.
Exemplary subsequences include Fab, Fab′, F(ab′)2, Fv, Fd, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv) and VL or VH, or other binding fragment of an intact immunoglobulin. Antibodies of the invention, useful in accordance with the invention methods, therefore include heavy-chain variable region sequence and light-chain variable region sequence of antibody that specifically bind B5R or H3L.
In further embodiments, an antibody binds to an antigenic region, determinant or epitope of B5R or H3L. Exemplary antigenic regions and epitopes of B5R or H3L homologs include, for example, a sequence region of B5R or H3L as set forth herein or known to one skilled in the art, or a subsequence or a portion thereof.
Antibodies of the invention further include one or more heterologous domains that impart a distinct function or activity on an antibody that binds B5R or H3L. Antibodies include an amino acid heterologous domain when one or more amino acids are distinct from the antibody (i.e., they are not a part of the native antibody). In one embodiment, a heterologous domain comprises a binding protein (e.g., receptor or ligand binding), an enzyme activity, a drug, an antiviral, a toxin, an immune-modulator, a detectable moiety or a tag.
Combination compositions including B5R and H3L binding antibodies, as well as methods of using such combinations, and methods in which such combinations are administered or combined with other compositions prior to, concurrently with or following administration of B5R or H3L antibody. In various embodiments, a composition includes antibody that binds B5R or H3L and an agent that decreases, reduces, inhibits, delays or prevents poxvirus infection or pathogenesis, replication, proliferation, or decreases, reduces, inhibits, delays or prevent the onset, progression, severity, frequency, duration or probability of one or more symptoms or complications associated with poxvirus (e.g., infectious or pathogenic poxvirus or vaccinia virus) infection or pathogenesis, or an adverse symptom or complication associated with vaccination or immunization with a vaccinia virus, vaccine virus protein, or a poxvirus or a poxvirus protein. Examples include a plurality (e.g., a pool) of monoclonal or polyclonal antibodies that each bind B5R and H3L, having the same or a different binding specificity or binding affinity, for B5R and H3L. An additional antibody that binds to a poxvirus protein, different from B5R or H3L binding antibody, can be administered separately from B5R or H3L binding antibody, or as a combination composition. In specific aspects, the additional antibody that binds to a poxvirus protein binds to IMV, cell-associated enveloped virion (CEV) or extracellular enveloped virion (EEV) forms of smallpox. In more specific aspects, the additional antibody binds to poxvirus protein B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, A2, or a B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, or A2 homolog. A plurality of antibodies can be individually administered or administered as a combination composition.
An additional composition may comprise VIG. Thus, compositions and methods of the invention include a combination composition of a B5R or H3L binding antibody and VIG, a combination composition of a B5R and H3L binding antibody and VIG, a combination method including administering separately or as a combination VIG with B5R or H3L binding antibody, and a combination composition of a B5R and H3L binding antibody and VIG.
Additional examples of a combination composition and combination method include administering separately or as a combination composition a protein with B5R or H3L binding antibody. In one specific aspect, a composition of B5R or H3L binding antibody includes an additional protein (e.g., an infectious or pathogenic poxvirus or vaccinia virus or vaccinia virus protein). In another specific aspect, a method includes administering an additional poxvirus or vaccinia virus protein prior to, concurrently with or following administration of B5R or H3L binding antibody. An additional poxvirus protein may be present on IMV, CEV or EEV forms of smallpox. In specific aspects, an additional poxvirus protein is one or more of B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, A2, or a B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, or A2 homolog.
Pharmaceutical compositions including antibodies of the invention and a pharmaceutically acceptable carrier or excipient are provided. Antibodies can be included in a pharmaceutically acceptable carrier or excipient prior to administration to a subject. Pharmaceutical compositions can be administered to a subject by systemic, regional or local delivery. In one embodiment, a method or carrier is suitable for administration systemically, regionally, or locally to a subject.
Kits that include one or more antibodies of the invention are also provided. In one embodiment, a kit includes instructions for treating (prophylaxis or therapeutic) one or more symptoms or complications associated with poxvirus infection of a subject by one or more poxvirus strains or isolates or species (e.g., virus infectivity, replication, proliferation, titer, onset, progression, severity, frequency, duration or probability of one or more symptoms as set forth herein or known in the art, etc.)
The invention is based at least in part on antibodies and subsequences thereof that specifically bind to poxvirus B5R envelope protein or B5R protein homologs. The invention is also based at least in part on antibodies and subsequences thereof that specifically bind to poxvirus H3L protein or H3L protein homologs. The invention is further based at least in part on combinations of such antibodies and subsequences.
Invention antibodies, among other things, can provide passive protection against an infectious vaccinia virus, and in multiple mouse models protected the animals from a lethal dose challenge of vaccinia virus. Antibodies of the invention are therefore useful for prophylactic (prior to poxvirus infection) and therapeutic (following poxvirus infection) treatment. In addition, antibodies of the invention are useful for prophylactic (prior to poxvirus infection) and therapeutic (following poxvirus infection) treatment in which subjects are at risk of an adverse side effect or complication associated with or caused by vaccination with a vaccinia virus (e.g., such as the live-virus preparation of vaccinia virus prepared from calf lymph, known as Dryvax®) or immunization against a poxvirus, for example, due to immune suppression. In addition, as inventionantibodies include humanantibodies, which are less likely to induce hypersensitivity from repeated administration and are more likely to remain in a human subjects' body for a longer period of time, antibodies of the invention can be administered to a human subject in advance of contact with or exposure to poxvirus (e.g., infectious or a pathogenic poxvirus such as smallpox), or a live or attenuated vaccinia virus, such as the live-virus preparation of vaccinia virus prepared from calf lymph (Dryvax®).
In accordance with the invention, there are provided antibodies and subsequences thereof that specifically bind to poxvirus B5R envelope protein. In one embodiment, anantibody or subsequence thereof that specifically binds to poxvirus B5R envelope protein includes a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any heavy chain variable region sequence set forth as SEQ ID NOs:2, 6, or 12, and a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any light chain variable region sequence set forth as SEQ ID NOs:4, 8 or 10. In another embodiment, anantibody or subsequence thereof that specifically binds to poxvirus B5R envelope protein, includes any heavy chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12, and any light chain variable region sequence set forth as SEQ ID NOs:4, 8 or 10, wherein the antibody or subsequence has one or more amino acid additions, deletions or substitutions of SEQ ID NOs:2, 6 or 12, or SEQ ID NOs:4, 8 or 10. In particular aspects, a sequence is at least 80-85%, 85-90%, 90-95%, 95-100% identical to any heavy chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12, or light chain variable region sequence set forth as SEQ ID NOs:4, 8 or 10. In further aspects, an antibody that specifically bind to poxvirus B5R envelope protein includes or consists of any one of a heavy chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12, or a light chain variable region sequence set forth as SEQ ID NOs:4, 8 or 10.
In accordance with the invention, there are also provided antibodies and subsequences thereof that specifically bind to poxvirus H3L envelope protein. In one embodiment, anantibody or subsequence thereof that specifically binds to poxvirus H3L envelope protein includes a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any heavy chain variable region sequence set forth as SEQ ID NO: 14, and a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any light chain variable region sequence set forth as SEQ ID NO:16. In another embodiment, anantibody or subsequence thereof that specifically binds topoxvirus H3L envelope protein includes any heavy chain variable region sequence set forth as SEQ ID NOs14 and any light chain variable region sequence set forth as SEQ ID NO:16, wherein the antibody or subsequence has one or more amino acid additions, deletions or substitutions of SEQ ID NO:14, or SEQ ID NO:16. In particular aspects, a sequence is at least 80-85%, 85-90%, 90-95%, 95-100% identical to any heavy chain variable region sequence set forth as SEQ ID NO:14, or any light chain variable region sequence set forth as SEQ ID NO:16. In further aspects, an antibody that specifically bind to poxvirus H3L envelope protein includes or consists of any one of a heavy chain variable region sequence set forth as SEQ ID NO:14, or a light chain variable region sequence set forth as SEQ ID NO:16.
The term “antibody” refers to a protein that binds to other molecules (antigens) via heavy and light chain variable domains, VH and VL, respectively. “Antibody” refers to any polyclonal or monoclonal immunoglobulin molecule, or mixtures thereof, such as IgM, IgG, IgA, IgE, IgD. Invention antibodies belong to any antibody class or subclass. Exemplary subclasses for IgG are IgG1, IgG2, IgG3 and IgG4.
As used herein, the term “monoclonal,” when used in reference to an antibody, refers to an antibody that is based upon, obtained from or derived from a single clone, including any eukaryotic, prokaryotic, or phage clone. A “monoclonal” antibody is therefore defined herein structurally, and not the method by which it is produced.
The terms “B5R antibody” or “anti-B5R antibody,” and grammatical variations thereof, mean a polyclonal or monoclonal antibody that binds to vaccinia virus extracellular enveloped virion (EEV) B5R protein (also referred to as “B5”), or a B5R protein homolog from a related poxvirus, such as monkeypox B5R or variola B6. Antibodies include specific or selective binding to B5R protein or a B5R protein homolog, which is selective for an epitope or antigenic determinant present in B5R protein or B5R protein homolog. That is, binding to proteins other than B5R or B5R protein homolog is such that the binding does not significantly interfere with detection of B5R or B5R protein homolog, unless such other proteins have significant similarity or the same epitope as epitope in B5R protein or B5R protein homolog that is recognized by a B5R homolog antibody. For example, anti-B5R antibodies 131C12, 131C14 and 131C18 also bind to poxvirus envelope protein 86, due to a conserved epitope shared by B5R and B6 proteins. Selective binding can be distinguished from non-selective binding using specificity, affinity, and competitive and non-competitive binding assays, described herein or known in the art.
The terms “H3L antibody” or “anti-H3L antibody,” and grammatical variations thereof, mean a polyclonal or monoclonal antibody that binds to vaccinia virus intracellular mature virion (IMV) H3L protein (also referred to as “H3”), or an H3L protein homolog. Antibodies include specific or selective binding to H3L protein or an H3L protein homolog, which is selective for an epitope or antigenic determinant present in H3L protein or H3L protein homolog. That is, binding to proteins other than H3L or H3L protein homolog is such that the binding does not significantly interfere with detection of H3L or H3L protein homolog, unless such other proteins have a similar or same epitope as epitope in H3L protein or H3L protein homolog that is recognized by an H3L/H3L homolog antibody. Selective binding can be distinguished from non-selective binding using specificity, affinity and other binding assays, and competitive and non-competitive binding assays, described herein or known in the art.
The term “isolated,” when used as a modifier of an invention composition (e.g., antibodies, subsequences, modified forms, nucleic acids encoding same, etc.), means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. The term “isolated” does not exclude alternative physical forms of the composition, such as multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.
An “isolated” composition (e.g., an antibody) can also be “substantially pure” or “purified” when free of most or all of the materials with which it typically associates with in nature. Thus, an isolated antibody that also is substantially pure or purified does not include polypeptides or polynucleotides present among millions of other sequences, such as antibodies of an antibody library or nucleic acids in a genomic or cDNA library, for example. A “substantially pure” or “purified” composition can be combined with one or more other molecules. Thus, “substantially pure” or “purified” does not exclude combinations of compositions, such as combinations of B5R and H3L antibodies, and other poxvirus antibodies or therapies.
Antibodies can be modified. Examples of modifications include one or more amino acid substitutions (e.g., 1-3, 3-5, 5-10 or more residues), additions or deletions (e.g., subsequences or fragments) of the antibody. In particular embodiments, a modified antibody retains at least part of a function or an activity of unmodified antibody, e.g., binding affinity (e.g., Kd) or binding specificity to B5R or H3L, binding to a vaccinia virus or pox virus in vitro or an infected cell (e.g., in culture), in vitro virus neutralization, complement-dependent virus neutralization, comettail inhibition, protection from or decreasing susceptibility to poxvirus infection or pathogenesis, or decreasing or preventing an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus or against poxvirus, etc.
A particular example of a modification is where an antibody is altered to have a different isotype or subclass by, for example, substitution of the heavy chain constant region. An alteration of Ig subclass can result in a change or an improvement in a function or activity (e.g., an anti-poxvirus activity, complement fixation, etc.). Thus, modifications include deleting small and large regions of amino acid sequences from an antibody and substituting the deleted region with another amino acid sequence, whether the sequence is greater or shorter in length than the deleted region.
Antibodies and subsequences of the invention include those having at least partial sequence identity to any heavy or light chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12 or SEQ ID NOs:4, 8 or 10, or SEQ ID NO:14, or SEQ ID NO:16. The percent identity of such antibodies and subsequences can be as little as 60%, or can be more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.). The percent identity can extend over the entire sequence length of a heavy or light chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12 or SEQ ID NOs:4, 8 or 10, or SEQ ID NO:14, or SEQ ID NO:16, or a contiguous region or area within any of SEQ ID NOs:2, 6 or 12 or SEQ ID NOs:4, 8 or 10, or SEQ ID NO:14, or SEQ ID NO:16. In particular aspects, the length of the sequence sharing the percent identity is 5 or more contiguous amino acids, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. contiguous amino acids. In additional particular aspects, the length of the sequence sharing the percent identity is 20 or more contiguous amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, etc. contiguous amino acids. In further particular aspects, the length of the sequence sharing the percent identity is 35 or more contiguous amino acids, e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 47, 48, 49, 50, etc., contiguous amino acids. In yet further particular aspects, the length of the sequence sharing the percent identity is 50 or more contiguous amino acids, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110, etc. contiguous amino acids.
The term “identity” and grammatical variations thereof, mean that two or more referenced entities are the same. Thus, where two antibody sequences are identical, they have the same amino acid sequence. The identity can be over a defined area (region or domain) of the sequence. “Areas, regions or domains” of homology or identity mean that a portion of two or more referenced entities share homology or are the same. Thus, where two antibody sequences are identical over one or more sequence regions they share identity in these regions.
The extent of identity between two sequences can be ascertained using a computer program and mathematical algorithm known in the art. Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region or area. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has exemplary search parameters as follows: Mismatch −2; gap open 5; gap extension 2. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).
In accordance with the invention, there are provided antibodies and subsequences in which there are one or more amino acid substitutions of any heavy or light chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12 or SEQ ID NOs: 4, 8 or 10, or SEQ ID NO:14, or SEQ ID NO:16. In particular embodiments, a heavy or light chain CDR (CDR1, CDR2 or CDR3) or FR will have 1-8, 1-5, 1-3 or fewer (e.g., 1 or 2) amino acid substitutions. In an additional embodiment, a substitution within a variable region sequence is not within a CDR. In another embodiment, a substitution within a variable region sequence is not within an FR. Exemplary heavy chain and light chain CDR sequences are as set forth in Table 2 (SEQ ID NOs:17-40): SSAMS; VISISGGSTYYADSVKG; ETRYYYSYGMDV; SYSMN; SISSSRSFIYYADSVKG; ERRYYYSYGLDV; SYSMN; SISSSSSYIYYADSVKG; ERRYYYSYGMDV; DYAIH; GISWNGRSIGYADSVKG; DIGFYGSGSLDY; RASQRIGFALA; DASSLET; QQFNTYPFT; RASQGISSALA; DASSLES; QQFNSYPYT; RASQGISSALA; DASSLES; QQFNSYPYT; RASQSVSSYLA; DASNRAT; and QQRSNWPALT.
The structural determinants that contribute to antigen (e.g., B5R and H3L) binding, such as complementarity determining regions (CDR) and framework regions (FR) within hypervariable regions are known in the art. The location of additional regions, such as D- and J-regions are also known. Antibodies and subsequences thereof in which one or more CDR and FR sequences will typically have sufficient sequence identity to a heavy or light chain sequence exemplified herein so as to retain at least partial function or activity of an antibody that includes a heavy and a light chain sequence exemplified herein, e.g., binding affinity (e.g., Kd) or binding specificity to B5R or H3L, binding to a vaccinia virus or pox virus or an infected cell in vitro (e.g., in culture), in vitro virus neutralization, complement-dependent virus neutralization, comet-tail inhibition, protection from or decreasing susceptibility to poxvirus infection or pathogenesis, or decreasing or preventing an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus or against poxvirus.
Amino acid substitutions can be conservative or non-conservative and may be in the constant or variable (e.g., hypervariable, such as CDR or FR) region of the antibody. One or a few amino acid substitutions (e.g., 2, 3, 4 or 5) in constant or variable regions are likely to be tolerated. Non-conservative substitution of multiple amino acids in hypervariable regions is likely to affect binding activity, specificity or antibody function or activity.
A “conservative substitution” means the replacement of one amino acid by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution is compatible with biological activity, e.g., specifically binds to B5R or H3L. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or similar size. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Particular non-limiting examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, and the like.
Amino acid substitutions may be with the same amino acid, except that a naturally occurring L-amino acid is substituted with a D-form amino acid. Modifications therefore include one or more D-amino acids substituted for L-amino acids, or mixtures of D-amino acids substituted for L-amino acids. Modifications further include structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms.
Regional mutability analysis can be used to predict the effect of particular substitutions in complementarity determining regions (CDR) and framework regions (FR) (Shapiro et al., J. Immunol. 163:259 (1999)). In brief, sequence comparison indicates a hierarchy of mutability among di- and trinucleotide sequences located within Ig intronic DNA, which predicts regions that are more or less mutable. Quantitative structure-activity relationship (QSAR) can be used to identify the nature of the antibody recognition domain and, therefore, amino acids that participate in ligand binding. Predictive models based upon QSAR can in turn be used to predict the effect of substitutions (mutations). For example, the effect of mutations on the association and dissociation rate of an antibody interacting with its antigen has been used to construct quantitative predictive models for both kinetic (Ka and Kd) constants, which can in turn be used to predict the effect of other mutations on the antibody (De Genst et al., J Biol Chem. 277:29897 (2002)). The skilled artisan can therefore use such analysis to predict Fvs (scFv), disulfide-linked Fvs (sdFv) and Vt, or VH domain subsequence has substantially the same or has the same B5R or H3L binding affinity or B5R or H3L binding specificity, or one or more functions or activities of B5R or H3L antibody, such as an anti-poxvirus activity in vitro or in vivo (e.g., virus neutralization, complement-dependent virus neutralization, comet-tail inhibition, efficacy in providing a subject with some protection against posvirus infection or pathogenesis, protecting or decreasing susceptibility of a subject from poxvirus infection or pathogenesis of a cell in vitro, or decreasing or preventing an adverse side effect or complication associated with or caused by vaccination or immunization with or against vaccinia virus). The terms “functional subsequence” and “functional fragment” when referring to an antibody of the invention refers to a portion of an antibody that retains at least a part of one or more functions or activities as an intact reference antibody.
B5R and H3L binding antibody subsequences, including single-chain antibodies, can include all or a portion of heavy or light chain variable region(s) (e.g., CDR1, CDR2 or CDR3) alone or in combination with all or a portion of one or more of the following: hinge region, CH1, CH2, and CH3 domains. Also included are antigen-binding subsequences of any combination of heavy or light chain variable region(s) (e.g., CDR1, CDR2 or CDR3) with a hinge region, CH1, CH2, and CH3 domains.
B5R and H3L antibody subsequences (e.g., Fab, Fab′, F(ab′)2, Fd, scFv, sdFv and VL or VH) can be prepared by proteolytic hydrolysis of the antibody, for example, by pepsin or papain digestion of whole antibodies. Antibody fragments produced by enzymatic cleavage with pepsin provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent to produce 3.55 Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and the Fc fragment directly (see, e.g., U.S. Pat. Nos. 4,036,945 and 4,331,647; and Edelman et al., Methods Enymol. 1:422 (1967)). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic or chemical may also be used. Genetic techniques include expression of all or a part of the H3L/H3L homolog antibody gene into a host cell such as Cos cells or E. coli. The recombinant host cells synthesize intact or single antibody chain, such as scFv (see, e.g., Whitlow et al., In: Methods: A Companion to Methods in Enzymology 2:97 (1991), Bird et al., Science 242:423 (1988); and U.S. Pat. No. 4,946,778). Single-chain Fvs and antibodies can be produced as described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods Enzymol. 203:46 (1991); Shu et al., Proc. Natl. Acad. Sci. USA 90:7995 (1993); and Skerra et al., Science 240:1038 (1988).
Additional modifications of antibodies included in the invention are antibody additions (derivatives)/insertions. For example, an addition can be the covalent or non-covalent attachment of any type of molecule to the antibody. Specific examples of antibody additions and derivatives include glycosylation, acetylation, phosphorylation, amidation, formylation, ubiquitinatation, and derivatization by protecting/blocking groups and any of numerous chemical modifications.
Additions further include fusion (chimeric) polypeptide sequences, which is an amino acid sequence having one or more molecules not normally present in a reference native (wild type) sequence covalently attached to the sequence. A particular example is an amino acid sequence of another antibody to produce a multispecific antibody.
Another particular example of a modified antibody having an amino acid addition is one in which a second heterologous sequence, i.e., heterologous functional domain is attached (covalent or non-covalent binding) that confers a distinct or complementary function upon the antibody. Such sequences can be referred to as chimeric sequences. For example, an Fc region can be a chimera that includes portions of human IgG1 and IgG3 Fc regions, which provides the antibody with increased complement fixation as compared to an antibody with an IgG1 or IgG3 Fc. In another example, an amino acid tag such as T7 or polyhistidine can be attached to antibody in order to facilitate purification or detection of antigen or poxvirus(es). Yet another example is an antiviral attached to an antibody in order to target cells infected with poxvirus for killing, proliferation inhibition, replication inhibition, etc. Thus, in other embodiments the invention provides antibodies and a heterologous domain, wherein the domain confers a distinct function, i.e. a heterologous functional domain, on the antibody.
Heterologous functional domains are not restricted to amino acid residues. Thus, a heterologous functional domain can consist of any of a variety of different types of small or large functional moieties. Such moieties include nucleic acid, peptide, carbohydrate, lipid or small organic compounds, such as a drug (e.g., an antiviral), a metal (gold, silver), radioisotope.
Linkers, such as amino acid or peptidimimetic sequences may be inserted between the antibody sequence and the heterologous functional domain so that the two entities maintain, at least in part, a distinct function or activity. Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character which could promote or interact with either domain. Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. The length of the linker sequence may vary without significantly affecting a function or activity of the fusion protein (see, e.g., U.S. Pat. No. 6,087,329). Linkers further include chemical moieties and conjugating agents, such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST).
Additional examples of heterologous functional domains are detectable labels. Thus, in another embodiment, the invention provides B5R and H3L antibodies that are detectably labeled.
Specific examples of detectable labels include fluorophores, chromophores, radioactive isotopes (e.g., S35, P32, I125), electron-dense reagents, enzymes, ligands and receptors. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert a substrate such as 3,3-′,5,5-′-tetramethylbenzidine (TMB) to a blue pigment, which can be quantified. Ligands may bind other molecules such as biotin, which may bind avidin or streptavidin, and IgG, which can bind protein A.
Modifications further include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond. Polypeptides may be modified in vitro or in vivo, e.g., post-translationally modified to include, for example, sugar residues, phosphate groups, ubiquitin, fatty acids, lipids, etc.
It is understood that an invention antibody or subsequence may have multiple (e.g., two or more) variations, modifications or labels. For example, a monoclonal antibody may be coupled to biotin to detect its presence with avidin as well as labeled with I125 so that it provides a detectable signal. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered to be within the scope of the invention.
The term “human,” when used in reference to an antibody, means that the amino acid sequence of the antibody is fully human. A “human B5R antibody” or “human anti-B5R antibody” therefore refers to an antibody having human immunoglobulin amino acid sequences, i.e., human heavy and light chain variable and constant regions that specifically bind to B5R protein or B5R protein homolog. A “human H3L antibody” or “human anti-H3L antibody” therefore refers to an antibody having human immunoglobulin amino acid sequences, i.e., human heavy and light chain variable and constant regions that specifically bind to H3L protein or H3L protein homolog. That is, all of the antibody amino acids are human or can or do exist in a human antibody. Thus, for example, an antibody that is non-human may be made fully human by substituting the non-human amino acid residues with amino acid residues that can or do exist in a human antibody. Amino acid residues present in human antibodies, CDR region maps and human antibody consensus residues are known in the art (see, e.g., Kabat, Sequences of Proteins of Immunological Interest, 4th Ed. US Department of Health and Human Services. Public Health Service (1987); and Chothia and Lesk J. Mol. Biol. 186:651 (1987)). A consensus sequence of human VH subgroup III, based on a survey of 22 known human VH III sequences, and a consensus sequence of human VL kappa-chain subgroup I, based on a survey of 30 known human kappa I sequences is described in Padlan Mol. Immunol. 31:169 (1994); and Padlan Mol. Immunol. 28:489 (1991)). Human antibodies of the invention therefore include antibodies in which one or more amino acid residues have been substituted with one or more amino acids present in any other human antibody.
The term “humanized” when used in reference to an antibody, means that the amino acid sequence of the antibody has non-human amino acid residues (e.g., mouse, rat, goat, rabbit, non-human primate, etc.) of one or more determining regions (CDRs) that specifically bind to the desired antigen (e.g., H3L) in an acceptor human immunoglobulin molecule, and one or more human amino acid residues in the Fv framework region (FR), which are amino acid residues that flank the CDRs. Human framework region residues of the immunoglobulin can be replaced with corresponding non-human residues. Residues in the human framework regions can therefore be substituted with a corresponding residue from the non-human CDR donor antibody to alter, generally to improve, antigen affinity or specificity, for example. In addition, a humanized antibody may include residues, which are found neither in the human antibody nor in the donor CDR or framework sequences. For example, a framework substitution at a particular position that is not found in a human antibody or the donor non-human antibody may be predicted to improve binding affinity or specificity human antibody at that position. Antibody framework and CDR substitutions based upon molecular modeling are well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Pat. No. 5,585,089; and Riechmann et al., Nature 332:323 (1988)). Antibodies referred to as “primatized” in the art are within the meaning of “humanized” as used herein, except that the acceptor human immunoglobulin molecule and framework region amino acid residues may be any primate amino acid residue (e.g., ape, gibbon, gorilla, chimpanzees orangutan, macaque), in addition to any human residue.
As used herein, the term “chimeric” and grammatical variations thereof, when used in reference to an antibody, means that the amino acid sequence of the antibody contains one or more portions that are derived from, obtained or isolated from, or based upon two or more different species. That is, for example, a portion of the antibody may be human (e.g., a constant region) and another portion of the antibody may be non-human (e.g., a murine heavy or light chain variable region). Thus, a chimeric antibody is a molecule in which different portions of the antibody are of different species origins. Unlike a humanized antibody, a chimeric antibody can have the different species sequences in any region of the antibody.
As used herein, the terms “B5R,” “B5”, “B5R protein,” “B5 protein,” “B5R sequence,” “B5 sequence,” “B5R domain” and “B5 domain” refer to all or a portion of an B5R protein sequence (e.g., a subsequence such as an antigenic region or epitope) isolated from, based upon or present in any naturally occurring or artificially produced (e.g., genetically engineered) poxvirus strain or isolate or subtype or a species of poxvirus. Thus, the term B5R and the like include B5R sequence of vaccinia virus, or B5R homolog of variola major and variola minor small pox virus, or monkeypox, as well as naturally occurring variants produced by mutation during the virus life-cycle, produced in response to a selective pressure (e.g., drug therapy, expansion of host cell tropism or infectivity, etc.), as well as recombinantly or synthetically produced B5R sequences. A B5R homolog is a sequence having a significant sequence similarity or identitiyidentity to one or more exemplary vaccinia virus B5R protein sequence sequences set forth as SEQ ID NOs:41-52. Typical sequence identities of B5R homologs in other poxviruses are 90% or more. Sequence identities of B5R homologs may be less, however. B5R homologs may be referred to by a different name, due to the position of the coding sequence in the virus genome, which determines the name. SequencesRepresentative non-limiting sequences and the names of B5R homologs are known in the art. B5R and B5R homologs to which antibodies bind include sequences within amino acid sequences set forth as SEQ ID NOs:41-52:
As used herein, the terms “H3L,” “H3”, “H3L protein,” “H3 protein,” “H3L envelope protein,” “H3 envelope protein,” “H3L sequence,” “H3 sequence,” “H3L domain” and “H3 domain” refer to all or a portion of an H3L envelope protein sequence (e.g., a subsequence such as an antigenic region or epitope) isolated from, based upon or present in any naturally occurring or artificially produced (e.g., genetically engineered) poxvirus strain or isolate or subtype or a species of poxvirus. Thus, the term H3L and the like include H3L sequence of vaccinia virus, or H3L homolog I3L of variola major and variola minor small pox virus, as well as naturally occurring variants produced by mutation during the virus life-cycle, produced in response to a selective pressure (e.g., drug therapy, expansion of host cell tropism or infectivity, etc.), as well as recombinantly or synthetically produced H3L sequences. An H3L homolog is a sequence having a significant sequence similarity or identity to exemplary vaccinia virus H3L protein sequence set forth as SEQ ID NOs:53-65. Typical sequence identities of H3L homologs in other poxviruses are 90% or more. Sequence identities of H3L homologs may be less, however. For example, molluscum contagiosum gene MC084L is a VV H3L homolog that has 29% identity, and is 53% similar to H3L set forth as SEQ ID NO:31. H3L homologs also typically have a similar length to exemplary H3L protein sequence set forth as SEQ ID NO:31, usually a length of about 320-330 amino acids. H3L homologs may be referred to by a different name, due to the position of the coding sequence in the virus genome, which determines the name. Exemplary names for H3L homologs are H3L, I3L, J3L and MC084L. Other sequences and the names of H3L homologs are also known in the art. Representative non-limiting H3L and H3L homologs to which antibodies bind include sequences within amino acid sequences set forth as SEQ ID NOs:53-65:
Predicted epitopes for H3L comprise three sequences, denoted PE1, PE2 and PE3, (SEQ ID NOs:66-68, respectively; U.S. Pat. No. 7,393,533) are underlined and in bold text, within an amino acid sequence (SEQ ID NO:53):
Invention antibodies include antibodies having kappa or lambda light chain sequences, either full length as in naturally occurring antibodies, mixtures thereof (i.e., fusions of kappa and lambda chain sequences), and subsequences/fragments thereof. Naturally occurring antibody molecules contain two kappa and two lambda light chains. The primary difference between kappa and lambda light chainsisin the sequences of the constant region.
The term “bind,” or “binding,” when used in reference to an antibody, means that the antibody specifically binds to all or a part of an antigen (e.g., B5R, H3L, or B5R or H3L homolog). Thus, an antibody specifically binds to all or a part of sequence or an antigenic epitope present on B5R, H3L, or B5R or H3L homolog, but may also bind to other proteins should those proteins have the same or a similar epitope as B5R, H3L, or B5R or H3L homolog antigenic epitope. Antibodies that bind to the same sequence or epitope or a part of the epitope as an antibody that binds to B5R, H3L, or B5R or H3L homolog, can have more or less relative binding affinity or specificity for B5R, H3L, or B5R or H3L homolog, and are expressly included. For example, in particular embodiments, antibodies are provided that bind to both B5R and B6 proteins, due to a shared epitope on the B5R and B6 proteins.
A part of an antigenic epitope means a subsequence or a portion of the epitope. For example, if an epitope includes 8 contiguous amino acids, a subsequence and, therefore, a part of an epitope may be 7 or fewer amino acids within this 8 amino acid sequence epitope. In addition, if an epitope includes non-contiguous amino acid sequences, such as a 5 amino acid sequence and an 8 amino acid sequence which are not contiguous with each other, but form an epitope due to protein folding, a subsequence and, therefore, a part of an epitope may be either the 5 amino acid sequence or the 8 amino acid sequence alone.
Epitopes typically are short amino acid sequences, e.g. about five to 15 amino acids in length. Systematic techniques for identifying epitopes are also known in the art and are described, for example, in U.S. Pat. No. 4,708,871. Briefly, a set of overlapping oligopeptides derived from an antigen may be synthesized and bound to a solid phase array of pins, with a unique oligopeptide on each pin. The array of pins may comprise a 96-well microtiter plate, permitting one to assay all 96 oligopeptides simultaneously, e.g., for binding to an anti-H3L monoclonal antibody. Alternatively, phage display peptide library kits (New England BioLabs) are commercially available for epitope mapping. Using these methods, binding affinity for every possible subset of consecutive amino acids may be determined in order to identify the epitope that a particular antibody binds. Epitopes may also be identified by inference when epitope length peptide sequences are used to immunize animals from which antibodies that bind to the peptide sequence are obtained. Continuous epitopes can also be predicted using computer programs, such as BEPITOPE, known in the art (Odorico et al., J. Mol. Recognit. 16:20 (2003)).
Antibodies of the invention include B5R and H3L antibodies and subsequences thereof with more or less affinity for B5R or H3L than a reference antibody. For example, anantibody or subsequence thereof can have more or less affinity for B5R envelope protein and include a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any heavy chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12, and a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any light chain variable region sequence set forth as SEQ ID NOs:4, 8 or 10. Anantibody or subsequence thereof can have more or less affinity for H3L envelope protein and include a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any heavy chain variable region sequence set forth as SEQ ID NO:14, and a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any light chain variable region sequence set forth as SEQ ID NO:16.
Antibodies may have the same or substantially the same binding specificity as the exemplified antibodies and subsequences thereof. Thus, a given antibody (e.g., B5R or H3L) may inhibit or compete for binding of another antibody to B5R or H3L, for example, inhibiting binding by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or more of C12, C14, C18, B96, B116, B126 or D67 antibodies. A given B5R or H3L antibody may not detectably compete with or inhibit binding of another antibody to B5R or H3L where the antibodies bind to regions of B5R or H3L that do not interfere with each other. Accordingly, antibodies and subsequences thereof that have substantially the binding specificity as antibodies and subsequences thereof that include a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any heavy chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12, and a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any light chain variable region sequence set forth as SEQ ID NOs:4, 8 or 10 are included, as are antibodies and subsequences thereof that have substantially the binding specificity as antibodies and subsequences thereof that include a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any heavy chain variable region sequence set forth as SEQ ID NO:14, and a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any light chain variable region sequence set forth as SEQ ID NO:16 are included.
To obtain antibodies that have the same or similar binding specificity as another antibody, antibodies that compete for the binding of the antibody to a target antigen (e.g., B5R or H3L) are screened using a conventional competition binding assay. Screened antibodies can be characterized by any method known in the art, and affinity or specificity, determined by competitive binding, for example, blocking or binding inhibition assays. Antibodies that have the same or similar binding specificity are those that compete for binding to the target antigen (e.g., B5R, B6 or H3L). Because the binding affinity of antibodies may differ, the antibodies will vary in their ability to compete for binding to antigen and may provide greater or less effectiveness for treatment as compared to other antibodies or VIG.
Invention antibodies therefore include human, humanized and chimeric antibodies having the same or different binding affinity for B5R or H3L or homolog thereof and having the same or a different binding specificity for B5R or H3L or homolog thereof. For example, a B5R or homolog antibody of the invention may have an affinity greater or less than 2-5, 5-10, 10-100, 100-1000 or 1000-10,000 fold affinity or any numerical value or range or value within such ranges, as another B5R antibody, for example, of C12, C14, C18, B96, or B116 antibody. Likewise, an H3L or homolog antibody of the invention may have an affinity greater or less than 2-5, 5-10, 10-100, 100-1000 or 1000-10,000 fold affinity or any numerical value or range or value within such ranges, as another H3L antibody, for example, D67 antibody. Antibodies of the invention therefore include human, humanized and chimeric antibodies having the same or different binding affinity or the same or different binding specificity, or function or activity (e.g., anti-poxvirus activity), as human polyclonal H3L, B5R or H3L or B5R protein homolog binding antibodies, as set forth herein.
Exemplary antibody binding affinities for a target antigen (e.g., B5R or H3L) have a dissociation constant (Ka) less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, and 10−15 M. Typically, binding affinities (Kd) for B5R or H3L will be less than 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, or 10−12 M.
At least a part of binding affinity for a target antigen (e.g., B5R or H3L) can be when the antibody has less affinity for target antigen (e.g., B5R or H3L) than a reference antibody, e.g., 1-3-fold, 1-5-fold, 2-5 fold, 5-10-fold, 5-15-fold, 10-15-fold, 15-20-fold, 20-25-fold, 25-30-fold, 30-50-fold, 50-100 fold, 100-500-fold 500-1000-fold, 1000-5000-fold, or less (e.g., Kd) affinity, or any numerical value or range of values within such ranges, for example, of C12, C14, C18, B96, B116, B126, or D67 antibody. At least a part of binding affinity for target antigen (e.g., B5R or H3L) can be when the antibody has more affinity for the target antigen (e.g., B5R or H3L) than a reference antibody, e.g., 1-3-fold, 1-5-fold, 2-5 fold, 5-10-fold, 5-15-fold, 10-15-fold, 15-20-fold, 20-25-fold, 25-30-fold, 30-50-fold, 50-100 fold, 100-500-fold 500-1000-fold, 1000-5000-fold, or more (e.g., Kd) affinity, or any numerical value or range of values within such ranges, for example, of C12, C14, C18, B96, B116, B126, or D67 antibody.
Binding affinity can be determined by association (Ka) and dissociation (Kd) rate. Equilibrium affinity constant, K, is the ratio of Ka/Kd. An antibody having the same binding affinity as another antibody, means that the dissociation constant (Kd) of each antibody is within about 1 to 10 fold (1-10 fold greater affinity or 1-10 fold less affinity, or any numerical value or range or value within such ranges, than the reference antibody). An antibody having “substantially the same” binding affinity as another antibody, means that the dissociation constant (Kd) of each antibody is within about 10 to 1000 fold (10-1000 fold greater affinity or 1-1000 fold less affinity), for example, of C12, C14, C18, B96, B116, B126, or D67 antibody.
Association (Ka) and dissociation (Kd) rates can be measured using surface plasmon resonance (SPR) (Rich and Myszka, Curr. Opin. Biotechnol. 11:54 (2000); Englebienne, Analyst. 123:1599 (1998)). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (BiaCore 2000, Biacore AB, Upsala, Sweden; and Malmqvist, Biochem. Soc. Trans. 27:335 (1999)).
Antibodies include those that have at least a part of an “activity” or “function” as the reference antibody, for example, binding affinity (e.g., Kd), binding specificity, or protection from or decreasing susceptibility to, or decreasing or preventing an adverse side effect or complication associated with or caused by poxvirus infection or pathogenesis or vaccination or immunization with a vaccinia virus or against poxvirus. Thus, an antibody having an activity of a B5R or H3L binding antibody has at least a part of one or more activities of the B5R or H3L binding antibodies, such as anti-poxvirus activity in vivo or in vitro, complement fixation, destruction of VACV infected cells by complement, etc.
The term “at least a part” means that the antibody may have less activity but the antibody retains at least a measurable or detectable amount of the activity of the reference antibody, e.g., at least partial binding affinity for B5R or H3L, at least partial protection from or decreasing susceptibility to, or decreasing or preventing an adverse side effect or complication associated with or caused by poxvirus infection or pathogenesis or vaccination or immunization with a vaccinia virus, vaccinia virus protein, against poxvirus, etc. B5R or H3L antibodies having at least a part of one or more activities or functions of the B5R or H3L binding antibodies and subsequences thereof exemplified herein may also have a greater activity than a reference antibody, such as one or more of the B5R or H3L binding antibodies and subsequences thereof exemplified herein. Invention antibodies include antibodies having either or both of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) activities.
Antibodies having an activity or function of B5R or H3L binding antibodies can be identified through various methods disclosed herein or known in the art. For example, binding assays against B5R or H3L on plates or (ELISA), on cells (cell based ELISA), and specific inhibition of antibody binding to B5R or H3L can be used as a measure of binding specificity as well as affinity. Additional assays include in vitro binding of vaccinia or poxvirus infected cells, neutralization assays with poxvirus (e.g., vaccinia virus), complement-dependent virus neutralization assay, comet-tail inhibition, as well as in vivo animal protection and other assays as set forth in Examples 4 to 7 and 10 in order to ascertain and compare antibodies for the ability to provide animals with protection against or protect or decrease susceptibility of vaccinia virus infection or pathogenesis, etc.
Methods of producing B5R and H3L binding antibodies are disclosed herein or known in the art. B5R and H3L binding polyclonal antibodies can be obtained by affinity purification of B5R or H3L antibodies from vaccinia immune globulin (VIG) from vaccinia virus or from B5R, H3L or B5R or H3L homolog immunized animals. Human VIG can be used as a source for human polyclonal B5R and H3L antibodies.
B5R and H3L binding monoclonal antibodies can be generated using techniques including conventional hybridoma technology, recombinant, and phage display technologies, or a combination thereof (see U.S. Pat. Nos. 4,902,614, 4,543,439, and 4,411,993; see, also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988). B5R and H3L binding monoclonal antibodies can also be obtained by direct cloning of immunoglobulin sequences from animals, including primate or human subjects that have been exposed to a poxvirus, or vaccinated or immunized with live or attenuated vaccinia virus or poxvirus protein.
Specific hybridomas that produce antibodies of the invention have been deposited with ATCC. In particular, hybridoma cell line 131C14AA (also referred to as C14), which produces an anti-B5R antibody, was deposited on Sep. 25, 2007, and has a deposit designation of PTA-8654 (ATCC 110801 University Blvd., Manassas, Va. 20110-2209); hybridoma cell line 131C18 (also referred to as C18), which produces an anti-B5R antibody, was deposited on Aug. 22, 2007, and has a deposit designation of PTA-8562 (ATCC 110801 University Blvd., Manassas, Va. 20110-2209); hybridoma cell line 131C12AA (also referred to as C12), which produces an anti-B5R antibody, was deposited on Sep. 25, 2007, and has a deposit designation of PTA-8653 (ATCC 110801 University Blvd., Manassas, Va. 20110-2209); and hybridoma cell line 130D67 (also referred to as D67), which produces an anti-H3L antibody, was deposited on Aug. 22, 2007, and has a deposit designation of PTA-8564 (ATCC 110801 University Blvd., Manassas, Va. 20110-2209).
Animals may be immunized with B5R, H3L or B5R or H3L homologs, including mice, rabbits, rats, sheep, cows or steer, sheep, goats, pigs, horse, guinea pigs, and primates including humans, in order to obtain antibodies that bind to B5R, H3L or B5R or H3L homolog. Such animals include genetically modified non-human animals having human IgG gene loci (e.g., lambda or kappa light chain), which are capable of expressing human antibodies. Conventional hybridoma technology using splenocytes isolated from immunized animals that respond to the antigen and fused with myeloma cells can be used to obtain human monoclonal antibodies. A specific non-limiting example is the human transchromosomic KM Mice™ (Tomizuka et al., Proc. Natl. Acad. Sci. USA 97:722 (2000); and Ishida et al., Cloning Stem Cells 4:91 (2004)) which can produce human immunoglobulin genes (WO02/43478) or HAC mice (WO02/092812). Transgenic animals with one or more human immunoglobulin genes (kappa or lambda) that do not express endogenous immunoglobulins are described, for example in, U.S. Pat. No. 5,939,598. Such animals can therefore be used to produce human antibodies in accordance with the invention compositions and methods. Additional methods for producing human polyclonal antibodies and human monoclonal antibodies are described (see, e.g., Kuroiwa et al., Nat. Biotechnol. 20:889 (2002); WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598). An overview of the technology for producing human antibodies is described in Lonberg and Huszar (Int. Rev. Immunol. 13:65 (1995)).
Antigen (e.g., B5R or H3L) suitable for generating antibodies can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. For example, B5R or H3L or subsequences thereof can be produced by standard peptide synthesis techniques, such as solid-phase synthesis. A portion of the protein may contain an amino acid sequence such as a T7 tag or polyhistidine sequence to facilitate purification of expressed or synthesized B5R or H3L sequence. B5R or H3L encoding nucleic acid may be expressed in a cell and protein produced by the cells purified or isolated. B5R or H3L may be expressed as a part of a larger protein by recombinant methods.
Forms of antigen (e.g., B5R and H3L) suitable for generating an immune response include peptide subsequences of full length antigen (e.g., B5R and H3L), which typically comprise four to five or more amino acids. Additional forms of antigen (e.g., B5R and H3L) include preparations or extracts (such as live or attenuated vaccinia virus, e.g., modified vaccinia Ankara (MVA), vaccinia virus Lister strain, vaccinia virus LC16m8 strain, vaccinia virus NYCBOH strain, vaccinia virus Wyeth strain or vaccinia virus prepared from calf lymph, Dryvax®), partially purified antigen (e.g., B5R and H3L) as well as host cells or viruses that express antigen (e.g., B5R and H3L) and preparations or mixtures of such antigen (e.g., B5R and H3L) expressing cells or viruses.
To increase the immune response, antigen (e.g., B5R and H3L) can be coupled to another protein such as ovalbumin or keyhole limpet hemocyanin (KLH), thyroglobulin and tetanus toxoid, or mixed with an adjuvant such as Freund's complete or incomplete adjuvant. Initial and any optional subsequent immunization may be through intraperitoneal, intramuscular, intraocular, or subcutaneous routes. Subsequent immunizations may be at the same or at different concentrations of antigen (e.g., B5R and H3L) preparation, and may be at regular or irregular intervals.
Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunol. 28:489 (1991); Studnicka et al., Protein Engineering 7:805 (1994); Roguska. et al., Proc. Nat'l. Acad. Sci. USA 91:969 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Human consensus sequences (Padlan, Mol. Immunol. 31:169 (1994); and Padlan, Mol. Immunol. 28:489 (1991)) have previously used to humanize antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993)).
Methods for producing chimeric antibodies are known in the art (e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191 (1989); and U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397). Chimeric antibodies in which a variable domain from an antibody of one species is substituted for the variable domain of another species are described, for example, in Munro, Nature 312:597 (1984); Neuberger et al., Nature 312:604 (1984); Sharon et al., Nature 309:364 (1984); Morrison et al., Proc. Nat'l. Acad. Sci. USA 81:6851 (1984); Boulianne et al., Nature 312:643 (1984); Capon et al., Nature 337:525 (1989); and Traunecker et al., Nature 339:68 (1989).
The invention also provides nucleic acids encoding B5R or H3L binding antibodies. In one embodiment, a nucleic acid encodes a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any heavy chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12 or a sequence set forth as SEQ ID NOs:4, 8 or 10. In another embodiment, a nucleic acid encodes a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to any heavy chain variable region sequence set forth as SEQ ID NOs:2, 6 or 12 or a sequence set forth as SEQ ID NOs:4, 8 or 10. In an additional embodiment, a nucleic acid encodes a sequence having one or more amino acid additions, deletions or substitutions of SEQ ID NOs:2, 6 or 12, or SEQ ID NOs:4, 8 or 10. In a further embodiment, a nucleic acid encodes a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to a sequence having one or more amino acid additions, deletions or substitutions of SEQ ID NO:14, or SEQ ID NO:16. In yet another embodiment, a nucleic acid encodes a sequence having one or more amino acid additions, deletions or substitutions of SEQ ID NO:14, or SEQ ID NO:16. In particular aspects, a nucleic acid encodes SEQ ID NOs:2, 6 or 12; or SEQ ID NOs:4, 8 or 10; SEQ ID NO:14; or SEQ ID NO:16, and subsequences thereof.
The terms “nucleic acid” and “polynucleotide” and the like refer to at least two or more ribo- or deoxy-ribonucleic acid base pairs (nucleotides) that are linked through a phosphoester bond or equivalent. Nucleic acids include polynucleotides and polynucleosides. Nucleic acids include single, double or triplex, circular or linear, molecules. Exemplary nucleic acids include but are not limited to: RNA, DNA, cDNA, genomic nucleic acid, naturally occurring and non naturally occurring nucleic acid, e.g., synthetic nucleic acid.
Nucleic acids can be of various lengths. Nucleic acid lengths typically range from about 20 nucleotides to 20 Kb, or any numerical value or range within or encompassing such lengths, 10 nucleotides to 10 Kb, 1 to 5 Kb or less, 1000 to about 500 nucleotides or less in length. Nucleic acids can also be shorter, for example, 100 to about 500 nucleotides, or from about 12 to 25, 25 to 50, 50 to 100, 100 to 250, or about 250 to 500 nucleotides in length, or any numerical value or range or value within or encompassing such lengths. In particular aspects, a nucleic acid sequence has a length from about 10-20, 20-30, 30-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-1000, 1000-2000, nucleotides, or any numerical value or range within or encompassing such lengths. Shorter polynucleotides are commonly referred to as “oligonucleotides” or “probes” of single- or double-stranded DNA. However, there is no upper limit to the length of such oligonucleotides.
Nucleic acids include sequences that are complementary and sequences that specifically hybridize to SEQ ID NOs:1, 3, 5, 7, 9, or 11; or SEQ ID NOs:13 or 15, or a complementary or antisense sequence thereof.
The term “complementary” or “antisense” refers to a polynucleotide or peptide nucleic acid capable of binding to a specific DNA or RNA sequence. Antisense includes single, double, triple or greater stranded RNA and DNA polynucleotides and peptide nucleic acids (PNAs) that bind RNA transcript or DNA. Particular examples include RNA and DNA antisense that binds to sense RNA. For example, a single stranded nucleic acid can target a protein transcript that participates in metabolism, catabolism, removal or degradation of glycogen from a cell (e.g., mRNA). Antisense molecules are typically 95-100% complementary to the sense strand but can be “partially” complementary, in which only some of the nucleotides bind to the sense molecule (less than 100% complementary, e.g., 95%, 90%, 80%, 70% and sometimes less), or any numerical value or range within or encompassing such percent values.
The term “hybridize” and grammatical variations thereof refer to the binding between nucleic acid sequences. Hybridizing sequences will generally have more than about 50% complementary to a nucleic acid that encodes an amino acid sequence of a reference (e.g., B5R or H3L heavy or light chain variable region) sequence. The hybridization region between hybridizing sequences typically is at least about 12-15 nucleotides, 15-20 nucleotides, 20-30 nucleotides, 30-50 nucleotides, 50-100 nucleotides, 100 to 200 nucleotides or more, or any numerical value or range within or encompassing such lengths.
Nucleic acid sequences further include nucleotide and nucleoside substitutions, additions and deletions, as well as derivatized forms and fusion/chimeric sequences (e.g., encoding recombinant polypeptide). For example, due to the degeneracy of the genetic code, nucleic acids include sequences and subsequences degenerate with respect to nucleic acids that encode SEQ ID NOs:2, 6 or 12; or SEQ ID NOs:4, 8 or 10; SEQ ID NO:14; or SEQ ID NO:16, and subsequences thereof.
Nucleic acids can be produced using various standard cloning and chemical synthesis techniques. Techniques include, but are not limited to nucleic acid amplification, e.g., polymerase chain reaction (PCR), with genomic DNA or cDNA targets using primers (e.g., a degenerate primer mixture) capable of annealing to antibody encoding sequence. Nucleic acids can also be produced by chemical synthesis (e.g., solid phase phosphoramidite synthesis) or transcription from a gene. The sequences produced can then be translated in vitro, or cloned into a plasmid and propagated and then expressed in a cell (e.g., a host cell such as eukaryote or mammalian cell, yeast or bacteria, in an animal or in a plant).
Nucleic acid may be inserted into a nucleic acid construct in which expression of the nucleic acid is influenced or regulated by an “expression control element.” An “expression control element” refers to a nucleic acid sequence element that regulates or influences expression of a nucleic acid sequence to which it is operatively linked. Expression control elements include, as appropriate, promoters, enhancers, transcription terminators, gene silencers, a start codon (e.g., ATG) in front of a protein-encoding gene, etc.
An expression control element operatively linked to a nucleic acid sequence controls transcription and, as appropriate, translation of the nucleic acid sequence. Expression control elements include elements that activate transcription constitutively, that are inducible (i.e., require an external signal for activation), or derepressible (i.e., require a signal to turn transcription off; when the signal is no longer present, transcription is activated or “derepressed”), or specific for cell-types or tissues (i.e., tissue-specific control elements).
Nucleic acid may be inserted into a plasmid for propagation into a host cell and for subsequent genetic manipulation. A plasmid is a nucleic acid that can be propagated in a host cell, plasmids may optionally contain expression control elements in order to drive expression of the nucleic acid encoding B5R or H3L binding antibody or antigen (e.g., B5R or H3L) in the host cell. A vector is used herein synonymously with a plasmid and may also include an expression control element for expression in a host cell (e.g., expression vector). Plasmids and vectors generally contain at least an origin of replication for propagation in a cell and a promoter. Plasmids and vectors are therefore useful for genetic manipulation and expression of B5R and H3L binding antibodies as well as antigen (e.g., B5R or H3L).
Nucleic acids encoding variable regions of B5R or H3L antibody heavy and light chains, or encoding full length B5R or H3L antibody heavy and light chains can be produced synthetically or using recombinant methods, or isolated from a cell such as a hybridoma. Isolated nucleic acids may be inserted into a suitable expression vector, and introduced into suitable host cells (e.g., CHO, plant and other cells) which can be cultured for the production of recombinant B5R or H3L antibodies.
In accordance with the invention, there are provided host cells that express or are transformed with a nucleic acid that encodes a B5R or H3L antibody of the invention. Host cells include but are not limited to prokaryotic and eukaryotic cells such as bacteria, fungi (yeast), plant, insect, and animal (e.g., mammalian, including primate and human) cells. For example, bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors; yeast transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus), or transformed animal cell systems engineered for stable expression.
The cells may be a primary cell isolate, cell culture (e.g., passaged, established or immortalized cell line), or part of a plurality of cells, or a tissue or organ ex viva or in a subject (in vivo). In particular embodiments, a cell is a hyperproliferative cell, a cell comprising a cellular hyperproliferative disorder, an immortalized cell, neoplastic cell, tumor cell or cancer cell.
The term “transformed” or “transfected” when use in reference to a cell (e.g., a host cell) or organism, means a genetic change in a cell following incorporation of an exogenous molecule, for example, a protein or nucleic acid (e.g., a transgene) into the cell. Thus, a “transfected” or “transformed” cell is a cell into which, or a progeny thereof in which an exogenous molecule has been introduced by the hand of man, for example, by recombinant DNA techniques.
The nucleic acid or protein can be stably or transiently transfected or transformed (expressed) in the cell and progeny thereof. The cell(s) can be propagated and the introduced protein expressed, or nucleic acid transcribed. A progeny of a transfected or transformed cell may not be identical to the parent cell, since there may be mutations that occur during replication.
Introduction of protein and nucleic acid into target cells (e.g., host cells) can also be carried out by methods known in the art such as osmotic shock (e.g., calcium phosphate), electroporation, microinjection, cell fusion, etc. Introduction of nucleic acid and polypeptide in vitro, ex vivo and in vivo can also be accomplished using other techniques. For example, a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers. A nucleic acid can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly (methylmethacrylate) microcapsules, respectively, or in a colloid system. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
Liposomes for introducing various compositions into cells are known in the art and include, for example, phosphatidylcholine, phosphatidylserine, lipofectin and DOTAP (e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282; and GIBCO-BRL, Gaithersburg, Md.). Piperazine based amphilic cationic lipids useful for gene therapy also are known (see, e.g., U.S. Pat. No. 5,861,397). Cationic lipid systems also are known (see, e.g., U.S. Pat. No. 5,459,127). Polymeric substances, microcapsules and colloidal dispersion systems such as liposomes are collectively referred to herein as “vesicles.”
Accordingly, viral and non-viral vector means of delivery into cells, tissue or organs, in vitro, in vivo and ex vivo are included.
In accordance with the invention, there are provided methods for providing a subject with protection against poxvirus infection or pathogenesis. In one embodiment, a method includes administering an amount of an antibody that binds to B5R or H3L envelope protein sufficient to provide the subject with protection against poxvirus infection or pathogenesis.
Also provided are methods for protecting or decreasing susceptibility of a subject to a poxvirus infection or pathogenesis. In one embodiment, a method includes administering an amount of an antibody that binds to B5R or H3L envelope protein sufficient to protect or decrease susceptibility of the subject to poxvirus infection or pathogenesis.
Additionally provided are methods for decreasing or preventing an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus, a vaccinia virus protein, a poxvirus or a poxvirus protein. In one embodiment, a method includes administering a composition comprising an amount of an antibody that binds B5R or H3L envelope protein to a subject sufficient to decrease or prevent an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus (live or attenuated) a vaccinia virus protein or a poxvirus or poxvirus protein. In various aspects, adverse side effects or complications decreased or prevented include adverse side effect or complication comprises: fever, rash, pustules or pocks, generalized vaccinia, progressive vaccinia, postvaccinial encephalitis, vaccinia keratitis, eczema vaccinatum, periocular infection or accidental infection of close contacts. In additional aspects, the subject is a candidate for or has been vaccinated with vaccinia virus, live or attenuated, a vaccinia virus protein, or a poxvirus or poxvirus protein. In other aspects, a subject is administered an antibody that binds B5R or H3L envelope protein or B5R or H3L envelope protein prior to, concurrently with, following or within 1-2, 2-4, 4-12 or 12-24 hours of vaccination or immunization with vaccinia virus, a vaccinia virus protein, or a poxvirus or poxvirus protein.
Further provided are methods for decreasing or preventing an adverse side effect or complication in an immune-suppressed subject (e.g., a subject with or at risk of immunodeficiency, such as an HIV-positive subject) associated with or caused by vaccination with a vaccinia virus, live or attenuated, or a vaccinia virus protein, or a poxvirus or poxvirus protein. In one embodiment, a method includes administering a composition comprising an amount of an antibody that binds B5R or H3L envelope protein to the subject sufficient to decrease or prevent an adverse side effect or complication associated with or caused by vaccination with a Vaccinia virus.
Methods of the invention may be practiced prior to, concurrently with, or following poxvirus infection, contact with or exposure to a poxvirus, or vaccination or immunization with a vaccinia virus, a vaccinia virus protein or a poxvirus or poxvirus protein. Methods of the invention may be practiced prior to, concurrently with, or following a poxvirus infection, contact with or exposure to a poxvirus, or vaccination with vaccinia virus, a vaccinia virus protein or immunization against a poxvirus or with a poxvirus protein.
Invention antibodies and methods include antibodies, subsequences thereof and methods that provide a subject with partial or complete protection against poxvirus infection or pathogenesis, or a reduction, inhibition, delay, decrease or prevention of a symptom of poxvirus exposure, infection or pathogenesis. Invention antibodies and methods include antibodies, subsequences thereof and methods that protect or decrease susceptibility of a subject, at least partially or completely, to a poxvirus infection or pathogenesis, or reduction, inhibition, delay, decrease or prevention of a symptom of pox virus infection or pathogenesis. Exemplary symptoms include, for example, high fever, fatigue, headache, backache, malaise, rash (maculopapular, vesicular or pustular) or lesions, viremia, delirium, vomiting, diarrhea, and excess bleeding. Antibody activity and methods of the invention can include any reduction, inhibition, delay, decrease or prevention in the onset, progression, severity, duration, frequency or probability of one or more symptoms associated with or caused by a poxvirus infection or pathogenesis, as set forth herein or known in the art, or a subjective or objective detectable or measurable improvement or benefit to the subject.
Invention antibodies and methods are applicable to vaccinia viruses and poxviruses generally, more specifically, members of the viral family Poxyiridae. Poxviruses can be infectious or pathogenic, or non-infectious or non-pathogenic. Specific non-limiting examples of pathogenic poxviruses include variola major and variola minor smallpox viruses. Additional specific non-limiting examples of pathogenic poxviruses include monkeypox, cowpox, Molluscum Contagiosum and camelpox. Vaccinia viruses are poxviruses that may be infectious or pathogenic, live or attenuated. Vaccinia viruses may be non-pathogenic, but may be infectious. Non-limiting examples of vaccinia virus express an H3L envelope protein. Typically, non-infectious live or attenuated vaccinia viruses are used to immunize human subjects against variola major and variola minor smallpox virus and related species of poxviruses. Examples of such vaccinia viruses include modified vaccinia Ankara (MVA), vaccinia virus Lister strain, vaccinia virus LC16m8 strain, vaccinia virus NYCBOH strain, vaccinia virus Wyeth strain or vaccinia virus Dryvax®. For example, VACVWR and VACVIHD-J are pathogenic in mice. Other non-limiting examples of Poxyiridae express homologs to H3L protein, which are proteins having significant sequence identity or similarity to H3L protein set forth as SEQ ID NO:31. H3L homologs can or are very likely to bind to an antibody that binds to H3L protein due to significant sequence identity or similarity.
Methods for treating poxvirus infection or pathogenesis of a subject, include administering to the subject an amount of an antibody that specifically binds B5R or H3L protein sufficient to treat poxvirus infection or pathogenesis. In various embodiments, a method provides a subject with protection against poxvirus infection or pathogenesis, protects or decreases susceptibility of a subject to poxvirus infection or pathogenesis, and decreases or prevents an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus or vaccinia virus protein, or a poxvirus or poxvirus protein. In the methods of the invention, antibody can be administered alone or in combination with other therapeutics (e.g., other antibodies, such as a B5R and H3L binding antibody combination, or VIG) prior to, concurrently with, or following, exposure to or contact with poxvirus, or poxvirus infection or pathogenesis. The antibody can be administered alone or in combination with other therapeutics (e.g., other antibodies, such as a B5R and H3L binding antibody combination, VIG or a poxvirus protein) prior to, concurrently with, or following, vaccination or immunization with a vaccinia virus, a vaccinia virus protein, or a poxvirus or poxvirus protein. Thus, prophylactic as well as therapeutic methods are provided.
Methods of the invention include methods in which treatment results in any beneficial effect, which is also considered therapeutic. Particular non-limiting examples of beneficial effects which are also considered therapeutic include reducing, decreasing, inhibiting, delaying or preventing poxvirus infection or pathogenesis, or poxvirus titer, proliferation or replication. Additional non-limiting particular examples of beneficial effects include reducing, decreasing, inhibiting, delaying, ameliorating or preventing onset, progression, severity, duration, frequency, probability or susceptibility of a subject to poxvirus infection or pathogenesis, one or more adverse symptoms or complications associated with poxvirus infection or pathogenesis, accelerating or facilitating or hastening recovery of a subject from poxvirus infection or pathogenesis or one or more symptoms thereof, or decreasing, preventing, reducing, inhibiting, or delaying an adverse side effect or complication associated with or caused by vaccination or immunization with a vaccinia virus, a vaccinia virus protein, a poxvirus, etc.
Methods of the invention therefore include providing a beneficial or therapeutic effect to a subject, for example, reducing, decreasing, inhibiting, delaying, ameliorating or preventing onset, progression, severity, duration, frequency or probability of one or more symptoms or complications associated with poxvirus infection or pathogenesis; reducing, decreasing, inhibiting, delaying or preventing increases in poxvirus titer, replication, proliferation, or an amount of a viral protein of one or more poxvirus strains or isolates or subtypes. Stabilizing the infection, pathogenesis, condition or symptom, or preventing or inhibiting or delaying a worsening or progression of the infection, pathogenesis, condition or a symptom or complication associated with poxvirus infection or pathogenesis, are also included in various embodiments of the methods of the invention.
Symptoms or complications associated with poxvirus infection and pathogenesis whose onset, progression, severity, frequency, duration or probability can be reduced, decreased inhibited, delayed ameliorated or prevented include, for example, high fever, fatigue, headache, backache, malaise, rash (maculopapular, vesicular or pustular) or lesions, delirium, vomiting, diarrhea, and excess bleeding. Other symptoms of poxvirus infection or pathogenesis, including variola major and variola minor smallpox virus, monkeypox, cowpox, molluscum contagiosum and camelpox, are known in the art and treatment thereof in accordance with the invention is provided.
In one embodiment, a method includes administering to the subject an amount of an antibody that specifically binds to B5R or H3L envelope protein sufficient to inhibit virus infection or pathogenesis. In additional embodiments, methods of the invention provide a subject with protection against poxvirus infection or pathogenesis, protect, reduce, decrease, inhibit or prevent susceptibility of a subject to pox virus infection or pathogenesis or one or more symptoms thereof, by one or more poxvirus strains or isolates or subtypes or a species of poxvirus. In particular aspects, antibody is administered prior to (prophylaxis), concurrently with or following poxvirus exposure or infection of the subject (therapeutic). Methods of the invention, in particular aspects, provide a beneficial or therapeutic effect which includes, for example, reducing or decreasing or delaying onset, progression, severity, frequency, duration or probability of one or more symptoms or complications of poxvirus infection or pathogenesis, virus titer, proliferation, replication or an amount of a viral protein of one or more poxvirus strains or isolates or subtypes or species, or susceptibility of a subject to infection or pathogenesis by one or more poxvirus strains or isolates or subtypes or species.
The methods of the invention, including treating poxvirus infection or pathogenesis or a symptom or complication associated with or caused by poxvirus infection or pathogenesis, or decreasing or preventing an adverse side effect or complication associated with or caused by immunization or vaccination with a vaccinia virus, vaccinia virus protein, or a poxvirus or poxvirus protein can therefore result in an improvement in the subjects' condition. An improvement can be any objective or subjective reduction, decrease, inhibition, delay, ameliorating or prevention of onset, progression, severity, duration, frequency or probability of one or more symptoms or complications associated with poxvirus infection or pathogenesis, or virus titer, replication, proliferation, or an amount of a viral protein, or an adverse side effect or complication associated with or caused by immunization or vaccination. An improvement can also be reducing or inhibiting or preventing increases in virus titer, replication, proliferation, or an amount of a viral protein of one or more poxvirus strains or isolates or subtypes or species. An improvement can also mean stabilizing the symptom or complication associated with poxvirus infection or pathogenesis, or inhibiting, decreasing, delaying or preventing a worsening or progression of the symptom or complication associated with poxvirus infection or pathogenesis, or progression of the underlying poxvirus infection. An improvement can therefore be, for example, in any of high fever, fatigue, headache, backache, malaise, rash (maculopapular, vesicular or pustular) or lesions, delirium, vomiting, diarrhea, or excess bleeding, to any degree or for any duration of time.
An improvement can but need not be complete ablation of any particular symptom or all symptoms, adverse side effects or complications associated with or caused by poxvirus exposure, contact, infection or pathogenesis or vaccinia virus, vaccinia virus protein or poxvirus or poxvirus protein immunization or vaccination. Rather, treatment may be any objective or subjective measurable or detectable improvement. For example, an improvement may reduce, delay or stabilize high fever, but may not reduce or stabilize fatigue, headache, backache, malaise, rash (maculopapular, vesicular or pustular) or lesions, delirium, vomiting, diarrhea, or excess bleeding. Thus, a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in the subject's condition or an associated symptom or complication, or an inhibition or prevention of worsening or progression of the symptom or condition (stabilizing the infection or pathogenesis, or one or more symptoms, adverse side effects or complications), over a short or long duration (hours, days, weeks, months, etc.).
An improvement also includes reducing or eliminating the need, dosage frequency or amount of an antiviral drug or other agent (e.g., protein, antibody) used for treating a subject having or at risk of having a poxvirus infection, or a symptom or complication associated with poxvirus infection.
Methods for protecting a subject from poxvirus infection, decreasing susceptibility of a subject to poxvirus infection or pathogenesis and accelerating or hastening a subject's recovery from poxvirus infection or pathogenesis by one or more poxvirus strains or isolates or subtypes or species are further provided. In one embodiment, a method includes administering to a subject having or at risk of having poxvirus infection or pathogenesis an amount of an antibody that specifically binds to B5R or H3L protein sufficient to protect the subject from poxvirus infection or pathogenesis, or to decrease susceptibility of the subject to poxvirus infection or pathogenesis. In another embodiment, a method includes administering to the subject an amount of antibody that specifically binds to B5R or H3L protein sufficient to accelerate or hasten a subject's recovery from poxvirus infection.
In invention methods in which improvement is a desired outcome, such as a prophylactic or therapeutic treatment method that provides an objective or subjective benefit as for poxvirus infection or pathogenesis, or an adverse side effect or complication associated with or caused by vaccination or immunization, an antibody can be administered in a sufficient or effective amount. As used herein, a “sufficient amount” or “effective amount” or an “amount sufficient” or an “amount effective” refers to an amount that provides, in single or multiple doses, alone or in combination with one or more other treatments, therapeutic regimens or agents (e.g., a drug), a long term or a short term detectable or measurable improvement or beneficial effect in a given subject of any degree or for any time period or duration (e.g., hours, days, months, years, or cured).
An amount sufficient or an amount effective can but need not be provided in a single administration and can but need not be achieved by a B5R or H3L antibody alone or in combination with each other or another compound, agent, treatment or therapeutic regimen. In addition, an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second or additional compound, agent, treatment or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional drugs, agents, treatment or therapeutic regimens may be included in order to provide a given subject with a detectable or measurable improvement or beneficial effect.
An amount sufficient or an amount effective need not be prophylactically or therapeutically effective in each and every subject treated, nor a majority of subjects treated in a given group or population. An amount sufficient or an amount effective means sufficiency or effectiveness in a particular subject, not a group or the general population. As is typical for such methods, some subjects will exhibit a greater or lesser response to a treatment method.
Methods of the invention, including, for example, prophylactic and therapeutic treatment methods, are applicable to any poxvirus strain or isolate or subtype or a species of poxvirus, or combination of strains or isolates or subtypes or species of poxviruses. Particular examples are infectious or pathogenic viruses that express B5R or H3L proteins or B5R or H3L homologs, such as a pox viruses expressing a sequence having sufficient sequence homology to B5R or H3L protein so as to bind to an antibody that binds to B5R or H3L protein. Specific non-limiting examples of poxviruses include variola major or variola minor smallpox virus. Additional specific non-limiting examples include monkeypox, cowpox, Molluscum Contagiosum, vaccinia and camelpox.
B5R and H3L antibodies of the invention may be combined with each other as well as other therapeutic agents. B5R and H3L antibodies of the invention may be administered as a combination with each other as well as other therapeutic agents in the methods of the invention. B5R and H3L antibodies of the invention may be administered alone prior to, concurrently with, or following administration with other therapeutic agents or treatment protocol or regimen, such as agents having anti-virus activity. Accordingly, combination compositions including B5R and H3L antibodies, methods of using such combinations, as well as methods in which other compositions are administered prior to, concurrently with or following administration of B5R or H3L antibody, in accordance with the methods of the invention, are provided.
Particular non-limiting examples of such combination compositions and combination methods include pooled monoclonal or pooled polyclonal antibodies containing two or more different antibodies that each bind B5R and H3L protein, having the same or a different binding specificity, binding affinity, or efficacy in inhibiting poxvirus infection of a cell in vitro or poxvirus infection or pathogenesis in vivo. In particular embodiments, a plurality of antibodies (e.g., B5R and H3L antibodies) are administered separately or as a combination composition in accordance with the invention. In further particular embodiments, an additional antibody that binds to a poxvirus protein, different from B5R or H3L binding antibody, is administered separately or as a combination composition with B5R and/or H3L binding antibody in accordance with the invention. In particular aspects, the additional antibody that binds to a poxvirus protein binds to one or more forms, for example, intracellular mature virion (IMV), cell-associated enveloped virion (CEV) or extracellular enveloped virion (EEV) forms of smallpox. In additional particular aspects, the additional antibody that binds to a poxvirus protein binds to vaccinia protein B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, A2, or a B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, or A2 homolog. In a further aspect, an additional antibody can include VIG.
Additional examples of such combination compositions and combination methods include administering separately or as a combination composition in accordance with the invention an additional poxvirus protein. In one particular embodiment, a B5R or H3L binding antibody includes an additional poxvirus protein. In another particular embodiment, a method includes administering an additional poxvirus protein. In particular aspects, the additional poxvirus protein is present on one or more of IMV, CEV or EEV forms of smallpox. In additional particular aspects, the additional poxvirus protein is one or more of vaccinia B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, A2, or a B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, or A2 homolog.
Compositions used in accordance with the invention, as well as methods, can exclude certain components or method steps. In one embodiment, a method in which the composition excludes one or more poxvirus proteins or one or more antibodies that bind to poxvirus proteins is administered. In particular aspects, a composition excludes or does not consist of live or attenuated vaccinia virus (e.g., modified vaccinia Ankara (MVA), vaccinia virus Lister strain, vaccinia virus LC16 m8 strain, vaccinia virus NYCBOH strain, vaccinia virus Wyeth strain, vaccinia virus ACAM2000 or vaccinia virus prepared from calf lymph, Dryvax®). In another embodiment, a method excludes administering one or more poxvirus proteins or one or more antibodies that bind to poxvirus proteins different from B5R or H3L protein. In particular aspects, a method excludes administering live or attenuated virus (e.g., poxvirus or modified vaccinia Ankara (MVA), vaccinia virus Lister strain, vaccinia virus LC16 m8 strain, vaccinia virus NYCBOH strain, vaccinia virus Wyeth strain or vaccinia virus prepared from calf lymph, Dryvax®) with an antibody that binds to B5R or H3L protein. In additional aspects, a method excludes administering poxvirus protein B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, A2, or a B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, or A2 homolog. In further aspects, a method excludes administering an antibody that binds to poxvirus protein. For example, human or non-human vaccinia immune globulin (VIG) or antibody that binds to B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, A2, or a B5R, L1R, D8L, A33R, A27L, A17L, L5, A21, H2, H3L, A28, A14, A56, A34, A36, or A2 homolog can be excluded in a method or composition that includes an antibody that binds to B5R or H3L protein.
Subjects appropriate for treatment include those having or at risk of having a poxvirus infection or pathogenesis or at risk of having a poxvirus infection. Target subjects therefore include subjects that have been exposed to or contacted with poxvirus, or that have developed one or more adverse symptoms of poxvirus infection or pathogenesis, regardless of the type, timing or degree of onset, progression, severity, frequency, duration of the symptoms.
Target subjects also include those at risk of poxvirus exposure, contact, infection or pathogenesis or at risk of having or developing any poxvirus infection or pathogenesis. The invention methods are therefore applicable to treating a subject who is at risk of poxvirus exposure, contact, infection or pathogenesis, but has not yet been exposed to or contacted with poxvirus. Prophylactic methods are therefore included. Target subjects for prophylaxis can be at increased risk (probability or susceptibility) of poxvirus exposure, contact, infection or pathogenesis, as set forth herein and known in the art. For example, a subject with acute or chronic immunological susceptibility (e.g., an immune-suppressed, immunocompromised, or HIV-positive subject) is at increased risk of poxvirus infection or pathogenesis.
“Prophylaxis” and grammatical variations thereof mean a method in which contact, administration or in vivo delivery to a subject is prior to contact with or exposure to poxvirus, or vaccination or immunization of a subject against poxvirus or with a vaccinia virus (e.g., an infectious or pathogenic poxvirus or live or attenuated vaccinia virus, or vaccinia Ankara (MVA), vaccinia virus Lister strain, vaccinia virus LC16m8 strain, vaccinia virus NYCBOH strain, vaccinia virus Wyeth strain or vaccinia virus prepared from calf lymph, Dryvax). In certain situations it may not be known that a subject has been contacted with or exposed to poxvirus, or vaccinated or immunized against poxvirus or with a vaccinia virus, but administration or in vivo delivery to a subject can be performed prior to manifestation or onset of poxvirus infection or pathogenesis (or an associated symptom). In either case, a method can eliminate, prevent, inhibit, decrease or reduce the probability of or susceptibility towards developing a symptom of poxvirus infection or pathogenesis, or an adverse side effect or complication associated with or caused by vaccination or immunization of a subject against poxvirus or with a vaccinia virus or a vaccinia virus protein or a poxvirus protein.
At risk subjects appropriate for treatment include subjects exposed to other subjects having any poxvirus. At risk subjects appropriate for treatment therefore include human subjects exposed to or at risk of exposure to other humans that may have a poxvirus infection, or are at risk of a poxvirus infection. At risk subjects appropriate for treatment also include subjects where the risk of poxvirus infection or pathogenesis is increased due to changes in virus infectivity or cell tropism, environmental factors, or immunological susceptibility (e.g., an immune-suppressed, immunocompromised, or HIV-positive subject).
Target subjects further include those at risk of an adverse side effect, complication or reaction associated with or caused by a smallpox vaccination (e.g., a live or attenuated vaccinia virus or a vaccinia virus protein, etc.) or immunization or treatment against small pox (e.g., vaccination or immunization with live or attenuated vaccinia virus or poxvirus, VIG, a vaccinia virus or a poxvirus protein, etc.). Such target subjects include those with atopic dermatitis. Subjects afflicted with atopic dermatitis are at risk of developing eczema vaccinatum when vaccinated or immunized against smallpox.
B5R and H3L antibodies and subsequences thereof can be administered in accordance with the methods as a single or multiple dose e.g., one or more times daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with poxvirus infection or pathogenesis, or an adverse side effect or complication associated with or caused by vaccination or immunization of a subject with a vaccinia virus, a vaccinia virus protein, or against a poxvirus or with a poxvirus protein.
Doses can vary depending upon whether the treatment is prophylactic or therapeutic, the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, the type of virus infection or pathogenesis to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a prophylactic or therapeutic benefit.
Typically, for therapeutic treatment, antibodies will be administered as soon as practical, typically within 1-2, 2-4, 4-12, 12-24 or 24-72 hours after a subject is exposed to or contacted with any poxvirus, or within 1-2, 2-4, 4-12, 12-24 or 24-48 hours after development of one or more symptoms associated with poxvirus infection or pathogenesis (e.g., onset of fever or rash) or symptoms associated with pathogenic poxviruses such as smallpox and monkeypox. For prophylactic treatment, antibodies can be administered 0-4 weeks, e.g., 2-3 weeks, prior to exposure to or contact with poxvirus since antibodies are predicted to be effective for at least a month following administration. For prophylactic treatment in connection with vaccination or immunization of a subject with vaccinia virus (live or attenuated), poxvirus or a poxvirus protein, antibodies can be administered prior to, concurrently with or following immunization of the subject. Typically, antibodies are administered concurrently with vaccination or immunization of a subject, but can be administered within 1-2, 2-4, 4-12, 12-24 or 24-48 hours prior to vaccination or immunization or within 1-2, 2-4, 4-12, 12-24 or 24-48 hours following vaccination or immunization.
Doses can be based upon current existing passive immunization protocols (e.g., VIG), empirically determined, determined using animal disease models or optionally in human clinical trials. Initial study doses can be based upon the animal studies set forth herein, for a mouse, which weighs about 20 (15-30) grams, and the amount of antibody administered that is determined to be effective. The dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1-10 μg/kg, 10-25 μg/kg, 25-50 μg/kg, 50-100 μg/kg, 100-500 μg/kg, 500-1,000 μg/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, two, three, four, or more times per hour, day, week, month or annually. A typical range will be from about 0.3 mg/kg to about 100 mg/kg.
The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by the status of the subject. For example, whether the subject has a poxvirus infection or pathogenesis, whether the subject has been exposed to or contacted with a poxvirus or is merely at risk of poxvirus contact or exposure, or whether the subject is a candidate for or will undergo vaccination or immunization with vaccinia virus (live or attenuated), poxvirus or a poxvirus protein. The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy.
The term “subject” refers to an animal, typically a mammalian animal, such as a non human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans. Subjects include animal disease models, for example, the mouse models of poxvirus infection (vaccinia virus) exemplified herein.
B5R and H3L binding antibodies of the invention, including modified forms, variants and subsequences/fragments thereof, and nucleic acids encoding B5R and H3L binding antibodies, can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for administration to a subject in vivo or ex vivo.
Antibodies can be included in a pharmaceutically acceptable carrier or excipient prior to administration to a subject. As used herein the term “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.
Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. Exemplary routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, ophthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, intralymphatic.
Formulations suitable for parenteral administration comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
For transmucosal or transdermal administration (e.g., topical contact), penetrants can be included in the pharmaceutical composition. Penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. For transdermal administration, the active ingredient can be formulated into aerosols, sprays, ointments, salves, gels, or creams as generally known in the art. For contact with skin, pharmaceutical compositions typically include ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils. Carriers which may be used include Vaseline, lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations thereof.
Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.
Supplementary active compounds (e.g., preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions may therefore include preservatives, anti-oxidants and antimicrobial agents.
Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation. Suitable preservatives are known in the art and include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.
An antimicrobial agent or compound directly or indirectly inhibits, reduces, delays, halts, eliminates, arrests, suppresses or prevents contamination by or growth, infectivity, replication, proliferation, reproduction, of a pathogenic or non-pathogenic microbial organism. Classes of antimicrobials include, antibacterial, antiviral, antifungal and antiparasitics. Antimicrobials include agents and compounds that kill or destroy (-cidal) or inhibit (-static) contamination by or growth, infectivity, replication, proliferation, reproduction of the microbial organism.
Exemplary antibacterials (antibiotics) include penicillins (e.g., penicillin G, ampicillin, methicillin, oxacillin, and amoxicillin), cephalosporins (e.g., cefadroxil, ceforanid, cefotaxime, and ceftriaxone), tetracyclines (e.g., doxycycline, chlortetracycline, minocycline, and tetracycline), aminoglycosides (e.g., amikacin, gentamycin, kanamycin, neomycin, streptomycin, netilmicin, paromomycin and tobramycin), macrolides (e.g., azithromycin, clarithromycin, and erythromycin), fluoroquinolones (e.g., ciprofloxacin, lomefloxacin, and norfloxacin), and other antibiotics including chloramphenicol, clindamycin, cycloserine, isoniazid, rifampin, vancomycin, aztreonam, clavulanic acid, imipenem, polymyxin, bacitracin, amphotericin and nystatin.
Particular non-limiting classes of anti-virals include reverse transcriptase inhibitors; protease inhibitors; thymidine kinase inhibitors; sugar or glycoprotein synthesis inhibitors; structural protein synthesis inhibitors; nucleoside analogues; and viral maturation inhibitors. Specific non-limiting examples of anti-virals include nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, zidovudine (AZT), stavudine (d4T), lamivudine (3TC), didanosine (DDI), zalcitabine (ddC), abacavir, acyclovir, penciclovir, valacyclovir, ganciclovir, 1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9->2-hydroxy-ethoxy methylguanine, adamantanamine, 5-iodo-2′-deoxyuridine, cidofivir, ST-246, trifluorothymidine, interferon and adenine arabinoside.
Pharmaceutical formulations and delivery systems appropriate for the compositions and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).
Antibodies and compositions thereof can be packaged in unit dosage form (capsules, troches, cachets, lozenges, or tablets) for ease of administration and uniformity of dosage. A “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms also include, for example, ampules and vials, which may include a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein. Individual unit dosage forms can be included in multi-dose kits or containers. Pharmaceutical formulations can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.
The invention provides kits comprising B5R and H3L antibodies, combination compositions and pharmaceutical formulations thereof, packaged into suitable packaging material. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., two or more B5R and H3L antibodies alone or in combination with an anti-poxvirus agent (e.g., a poxvirus protein or an antibody that binds to a poxvirus protein different than B5R and H3L, VIG, etc.) or drug.
The term “packaging material” refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).
Kits of the invention can include labels or inserts. Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., a diskette, hard disk, ZIP disk, etc.), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date.
Labels or inserts can include information on a condition, disorder or disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, treatment protocols or prophylactic or therapeutic regimes described herein. Exemplary instructions include, instructions for treating poxvirus infection or pathogenesis. Kits of the invention therefore can additionally include labels or instructions for practicing any of the methods of the invention described herein including treatment, detection, monitoring or diagnostic methods. Thus, for example, a kit can include an antibody that has one or more anti-poxvirus functions or activities as set forth herein, together with instructions for administering the antibody in a prophylactic or therapeutic treatment method of the invention.
Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.
Invention kits can additionally include a growth medium (e.g., for a B5R or H3L binding antibody producing cell line), buffering agent, or a preservative or a stabilizing agent in a pharmaceutical formulation containing a B5R or H3L binding antibody. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package. Invention kits can be designed for cold storage. Invention kits can further be designed to contain B5R or H3L binding antibody producing hybridoma or other host cells (e.g., CHO cells). The cells in the kit can be maintained under appropriate storage conditions until the cells are ready to be used. For example, a kit including one or more hybridoma or other cells can contain appropriate cell storage medium so that the cells can be thawed and grown.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.
As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a “B5R or B5R homolog binding antibody” or “an H3L or H3L homolog binding antibody” includes a plurality of such antibodies and reference to an “activity or function” such as “an anti-poxvirus activity or function” can include reference to one or more activities or functions, and so forth.
As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as a percentage range, 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Reference to a range of 1-5 fold therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.
The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. For example, in certain embodiments or aspects of the invention, antibodies or subsequences that specifically bind to poxvirus proteins are excluded. In certain embodiments and aspects of the invention, poxvirus proteins are excluded. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include embodiments and aspects that expressly exclude compositions (e.g., poxvirus antibodies or proteins) and method steps are nevertheless disclosed and included in the invention.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims.
EXAMPLES Example 1This example describes various materials and methods.
Antigen Preparation
Baculovirus expressed vaccinia B5R recombinant protein: DNA was isolated from 5×106 vaccinia virus infected Hela cells using the QIAamp DNA Mini Kit (QIAGEN Inc., Valencia, Calif.) following manufacturer's instructions. The sequence encoding the extracellular domain of vaccinia B5R was amplified by reverse-transcription polymerase chain reaction using primers B5R-Ftopo dir gate and B5R-Rtopo dir gate (Table 1). The product was sub-cloned into the pENTR™/D-TOPO® Gateway entry vector (Invitrogen Corp, Carlsbad, Calif.). The cloned PCR amplified product was sequenced and confirmed to be identical to the published sequence of vaccinia B5R WR stain (SEQ NO:28). Recombinant baculovirus were generated that encoded the C-terminal 6×His tagged vaccinia B5R protein (B5R-His) by performing the recombination reaction between the Gateway pENTR™/D-TOPO B5R-His and the Gateway BaculoDirect™ C-term linear DNA (Invitrogen Corp, Carlsbad, Calif.). Trichoplusia ni High-Five BTI-TN-5b1-4 (Tn5) insect cells (Invitrogen Corp.) were infected with the B5R-His recombinant baculovirus for protein production.
The nucleotide sequence of vaccinia B5R-6×His protein from initiation codon (ATG) to 6×His tag (bold) is as follows (SEQ ID NO:69):
The amino acid sequence of vaccinia virus B5R 6×His protein, signal peptide (bold) and 6×His (underlined) is as follows (SEQ ID NO:70):
Bacterially Expressed Recombinant H3L Protein:
The DNA from cells infected with the vaccinia virus that was used for B5R cloning above, was likewise used as a PCR template for cloning full length H3L. The sequence encoding the full-length open reading frame of vaccinia H3L was amplified by reverse-transcription polymerase chain reaction using primers H3L F NdeI pET-15b and H3L R Bam HI pET-15b (Table 1). As a cloning intermediate, this PCR product was cloned into the TA-topo 2.1 vector following manufacturers instructions (Invitrogen, Carlsbad Calif.). This H3L-TA-topo2.1 plasmid was then used as a template for PCR using primers H3L NheI fwd pET28 and H3L XhoI rev pET28 (Table 1). The amplified full-length PCR product was digested with NheI and XhoI and ligated into the NheI and XhoI sites of the bacterial expression vector pET28a to create pET28a-full length H3L-His, which encodes a H3L with a C-terminal 6×His tag.
The nucleotide sequence of vaccinia H3L-6×His protein from initiation codon (ATG) to 6×His tag (bold) is as follows (SEQ ID NO:71):
The amino acid sequence of vaccinia virus H3L-His protein and 6×His (underlined) is as follows (SEQ ID NO:72):
H3L production and protein purification: To produce H3L-His in bacteria, the expression vector pET28a-full length H3L-His was transformed into BL21 (DE3) pLysS competent cells and bacterial cultures were induced to express H3L-His by a 2 hr. incubation of diluted (1:20) overnight cultures in 1 mM IPTG (sopropyl-beta-D-thiogalactopyranoside). Cells were harvested by centrifugation for protein purification. Recombinant full-length H3L with N-terminal HisTag® was purified by metal chelate affinity chromatography with Ni Sepharose 6 Fast Flow resin (GE Healthcare). Bacterial cells were lysed with microfluidizer (model M10L, Microfluidics, Inc.). Lysis buffer included 0.5% Triton X-100 (Calbiochem), and the chromatography was performed in the presence of appropriate non-ionic detergent, like 0.5% Triton X-100 or 0.58% Octyl Glucoside (Calbiochem). H3L was eluted from the column with 200 mM imidazole, and subsequently dialyzed against 20 mM Na phosphate buffer, pH 8.0, 0.25M NaCl, 10% glycerol, and appropriate detergent (see above). Protein concentration was determined by DC Lowry protein assay (Bio-Rad) using BSA standard (Pierce Biotechnology) in the same buffer.
B5R production and protein purification: B5R-His recombinant protein was generated by infecting 1 liter of insect Tn5 cells with B5R-His baculovirus for 4 days. Growth media was harvested and clarified by centrifugation. Recombinant B5R-His was purified by metal chelate affinity chromatography with Ni Sepharose 6 Fast Flow resin (GE Healthcare). The baculovirus-infected Tn5 insect cell supernatant was concentrated and diafiltered into PBS by tangential flow filtration using a membrane with 10 kDa molecular weight cut off. B5R was eluted from the column with 200 mM imidazole, and subsequently dialyzed against PBS. Protein concentration was determined by Lowry protein assay (Bio-Rad) using BSA standard (Pierce Biotechnology).
Mice: Human trans-chromosomic KM Mice™ (WO02/43478, WO02/092812, Ishida and Lonberg, IBC's 11th Antibody Engineering Meeting. Abstract (2000); and Kataoka, S. IBC's 13th Antibody Engineering Meeting. Abstract (2002)) harboring human chromosome fragments encoding the human immunoglobulin region were obtained from Kirin Brewery Co., Ltd., Japan, and were housed in the animal facility at the La Jolla Institute for Allergy and Immunology. An overview of the technology for producing human antibodies is described in Lonberg and Huszar (Int. Rev. Immunol. 13:65 (1995)). Transgenic animals with one or more human immunoglobulin genes (kappa or lambda) that do not express endogenous immunoglobulins are described, for example in, U.S. Pat. No. 5,939,598. Additional methods for producing human antibodies and human monoclonal antibodies are described (see, e.g., WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598). Development of bovine carrying human immunoglobulin genes, TC cows, is described in Kuroiwa et al., Nat. Biotechnol. 20:889 (2002), and Kuroiwa et al., Nat. Genet. 36:775 (2004).
Immunization: B5R-His or H3L-His recombinant protein was mixed with an equal volume of complete Freund's adjuvant (CFA, Sigma) and an emulsion was prepared. KM Mice™ were immunized subcutaneously with 25 to 50 μg protein of soluble recombinant B5R-His or H3L-His in CFA/IFA. Mice were boosted subcutaneously with 10 to 20 μg of protein emulsified in incomplete Freund's adjuvant (IFA, Sigma) at 1 to 2 week intervals for 2 boosts. The first boost was with 10% CFA/90% IFA, the second boost was IFA alone. A final intravenous injection of 10 μg of soluble B5R-His or H3L-His without adjuvant was given 3 days prior to fusion.
Hybridoma production: Several of the mice raised anti-B5R or H3L specific antibodies, with a range in human IgG B5R or H3L specific titers. The mice with the highest anti-B5R or anti-H3L IgG specific antibody titer in their serum were selected for production of monoclonal antibodies. Spleens were harvested and single cell suspensions were fused to a myeloma cell line (SP2/O—Ag14) (ATCC, Rockville, Md.) at a ratio of 5:1 with 50% polyethylene glycol (Boehringer Mannheim, Indianapolis, Ind.) to generate human anti-vaccinia B5R or H3L producing hybridomas. The fusions were plated into 96 well flat, bottom plates at an optimal density and cultured in complete DMEM-10 medium (Dulbecco's Modified Engle's Medium with 10% fetal bovine serum (FBS, Invitrogen, Corp.), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate (all from BioWhittaker, Walkersville, Md.), HAT supplement (Sigma), and 10% Hybridoma Cloning Factor (HCF, Biovaris, San Diego, Calif.) in a 10% CO2, 37° C. incubator. Approximately 1100 wells from 4 fusions were screened by ELISA for human IgG containing B5R or H3L specific antibodies. Production of human anti-vaccinia B5R or H3L IgG antibodies were confirmed by ELISA. Crude hybridoma supernatant was used for a preliminary evaluation of virus neutralizing activity in vitro. Positive wells were expanded and subjected to 2 to 3 rounds of limiting dilution cloning to obtain monoclonal antibodies.
Antibody protein purification: For antibody purification, hybridomas were cultured in 2 liter roller bottles at 350 milliliter to 1 liter/bottle or in a 1 liter Integra system (INTEGRA Bioscience, Inc. Ijamsville, Md.) with hybridoma-SFM medium (Invitrogen, Corp.) supplemented with ultra low IgG fetal bovine serum (Invitrogen, Corp.) Human monoclonal antibodies were purified from culture media using recombinant Protein A-Sepharose Fast Flow gel (Amersham Biosciences). Conditioned medium generated in roller bottles was first concentrated using an Ultrasette tangential flow system (Pall Corp., East Hills, N.Y.). The conditioned medium was filtered with a 0.22 μm vacuum filter unit (Millipore, Bedford, Mass.) and loaded onto a Protein A-Sepharose Fast How column (Amersham Biosciences) of appropriate size for the amount of human antibody in the medium. The column was washed thoroughly with 20 column volumes of PBS and the antibody was eluted with 0.1 M Gly-HCl, pH 3.6, 0.15 M NaCl and neutralized with 1 M Tris-HCl, pH 8.0. The fractions were analyzed by SDS-PAGE and the positive fractions were pooled and concentrated with a centrifugal concentrator (Vivaspin, 50,000 MWCO: Sartorius, Gettingen, Germany). Sephadex G-25 desalting columns, (NAP, Amersham Biosciences), were used for buffer exchange to PBS, pH 7.4. Finally, the antibody was filter sterilized using syringe filters with 0.22 μm pore diameters and the antibody concentration was determined by the Lowry method. Pyrogen content was determined using a Limulus Amebocyte Lysate (LAL) assay (Associates of Cape Cod, Falmouth, Mass.). The limits of detection of this assay are 0.06 EU/mg. If the test was negative, the samples were considered endotoxin free.
Human IgG Quantitation ELISA: To determine the amount of human antibody present in supernatants and purified stocks the following protocol was used. Goat anti-human Fcγ specific antibody (Jackson Immunoresearch Laboratories, West Grove, Pa.) was coated to 96 well plates (Nunc, Denmark) in carbonate buffer at 0.5 μg/well for 1 hour at 37° C. The plates were then blocked with Superblock (Pierce, Rockford, Ill.) for 30 minutes followed by addition of the samples to the plates. Standard curves were generated using total human IgG (Sigma) or purified human IgG1 or IgG4 (Kirin Brewery Co., Ltd). The plates were incubated for 1 hour at 37° C., washed in PBS/1% BSA/0.1% Tween20 (Sigma), and the bound antibody was detected with goat anti-human Fcγ specific antibody conjugated to horseradish peroxidase (HRP, Jackson Immunoresearch) for 1 hour at 37° C. The TMB substrate (Sigma) was added for 10 minutes and the reaction was stopped with H2SO4 (LabChem, Pittsburgh, Pa.). The OD was measured at 450 nm on a microplate reader.
B5R or H3L Specific Antibody Detection ELISA: Antibody titers, specificity, and production by hybridomas were determined by ELISA. In brief, 96 well flat bottom plates were coated with 50 μl of B5R-His or H3L at 5 μg/ml in carbonate buffer (pH 9.4) overnight at 4° C. or at 37° C. for 1 hour. After washing twice with PBS/0.1% Tween 20, plates were blocked with PBS/1% BSA/0.1% Tween20 at 37° C. for 1 hour. The serum, supernatant, or purified antibody was diluted in blocking buffer, added to the wells, and the plates were incubated for 1 hour at 37° C. The plates were washed 4 times with PBS/0.1% Tween 20 and the peroxidase conjugated sheep anti-human kappa detection antibody (The Binding Site, Birmingham, UK) was added at a dilution of 1:2000. Following a 1 hour incubation at 37° C., the plates were washed and the TMB (Sigma) substrate was added and incubated at room temperature for 10 to 30 minutes. The reaction was stopped with H2SO4 (LabChem) and the optical density was measured at 450 nm by a microplate reader. ELISAs were also performed using whole vaccinia virus antigen from infected cell lysates.
Anti-B5R or H3L Antibody Cross-blocking (Binding Competition) Assays: In order to determine if the antibodies bind the same “epitope” of B5R or H3L an ELISA protocol was used. Nunc 96 well flat bottom ELISA plates were coated with the human anti-B5R or H3L antibodies in carbonate buffer at 2 μg/ml for 1 hour at 37° C. The plates were washed and then blocked with PBS/1% BSA/Tween 20. The human anti-B5R or H3L antibodies were then pre-incubated with recombinant B5R-His or H3L protein for 30 minutes at room temperature. The combinations of antibody-B5R or H3L protein were added to the plate and incubated for 1 hour at 37° C. After 3 washes, bound B5R-His or H3L-His was detected with peroxidase conjugated mouse anti-poly His IgG2a (Clonetech). The ELISA was completed as described above. The percent inhibition was determined using the OD of each sample in the following formula: % inhibition=100−((sample/Maximum binding)*100).
Isolation of Human Anti-B5R or H3L Antibody Genes: Cultured hybridoma cells, which produce either anti-B5R or H3L antibodies were collected by centrifugation. Total RNA was purified from these cells using the RNeasy kit (QIAGEN Inc., Valencia, Calif.) following the manufacturer's instructions. The SMART-RACE cDNA Amplification Kit (Clontech Co., Ltd., Palo Alto, Calif.) was used for cloning of cDNA that encodes the variable region of the immunoglobulin genes from total hybridoma cell RNA. Briefly, first strand cDNA was prepared by reverse transcriptase from 2 microgram of total RNA. This cDNA was used as a template for polymerase chain reaction (PCR) to amplify the variable region and a part of the constant region of heavy and light chains (HV and LV, respectively). The amplified sequences also contained the antibody leader peptide sequences. The reaction was as follows: 2.5 U Pfu Ultra DNA polymerase (Stratagem, La Jolla, Calif.); 0.2 μM 3′ Primer (for Heavy chain: IgG1p, for Light chain: hk5, Table 1); 1× Universal Primer Mix A for the 5′ end (UMP primer Mix A included in the SMART RACE Kit); 200 μM dNTP mix; 1 mM MgCl2; Pfu Ultra Buffer (final concentration is 1×); and cDNA template.
The thermocycling program was 5 cycles of: 94° C.×30 sec, 72° C.×3 min. 5 cycles of: 94° C.×30 sec, 70° C.×30 sec, 72° C.×3 min. 25 cycles of: 94° C.×30 sec, 68° C.×30 sec, 72° C.×3 min followed by an extension at 72° C.×7 min. Amplified DNA fragments were collected by agarose gel electrophoresis, and purified using the QIAquick Gel Extraction Kit (Qiagen Co., Ltd., Germany). Purified DNA fragments of HV and LV were integrated into the PCR 4 Blunt-TOPO vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen, Carlsbad, Calif.), and each construct plasmid was transformed into E. coli. Bacterial colonies, were selected and plasmid purified from those containing plasmid with HV or LV sequences. Nucleotide sequences of each insert (HV and LV) in the construct plasmids were analyzed using specific primers (M13F, M13R, Table 1). Based on the sequence obtained from HV and LV, oligonucleotide primers were designed to amplify VH and VL (Table 1).
Anti-B5R 131C12 antibody VH and VL were cloned into the IgG1 mammalian expression vector. Briefly, oligonucleotide primers, containing 5′-SalI and 3′-NheI restriction enzyme recognition sites were designed to amplify the variable region of the Heavy chain (HV) by PCR. PCR was performed using pTopo-VH miniprep DNA as a template and antibody clone 131C12 specific primers 12H FWD SalI and 12H REV NheI (Table 1), with Pfu Ultra DNA polymerase. After digestion of the PCR product with NheI and SalI, a 410 bp fragment was sub-cloned into the IgG1 expression vector (IDEC Pharmaceuticals, San Diego, Calif., N5KG1-Val Lark (a modified vector of N5KG1, U.S. Pat. No. 6,001,358)) that was pre-digested with NheI and SalI (8.9 kilobases DNA fragment). The existence of variable region of the Heavy chain (HV) was analyzed by restriction digest.
As the second step, LV was inserted into N5KG1-Val Lark-VH vector as follows: the DNA vector was digested by two DNA restriction enzymes, BglII and BsiWI. The 9.1 kb DNA fragment was isolated. Similarly to the heavy chain construct, a primer set for PCR of LV was designed to contain the recognition sites for 5′BglI and 3′BsiWi. These primers, 12K FWD BglII and 12K REV BsiWI (Table 1), were used to amplify LV from the pTopo-LV miniprep plasmid DNA. The PCR product was digested with BglII and BsiWI and isolated by agarose gel electrophoresis and gel purification. This fragment, containing either B5R or H3L specific antibody LV, was ligated to the prepared 9.1 kb vector with T4 DNA ligase and used to transform Top10 cells (Invitrogen). Positive E. coli transformants were selected. The resulting expression vectors were purified, and the presence of both LV and HV regions confirmed by restriction analysis and DNA sequencing.
Generation of vectors to produce recombinant 131C14, 131C18, 130D67 and 130D53 antibodies was performed in the manner described above, the 3′ primers used for amplification of the heavy and light chain genes in the RACE reactions were HH-2 and HK-2, respectively.
Amplification of the heavy chains for 131C14, 131C18, 130D25, 130D67 and 130D53 was performed using the respective primers listed in Table 1. The 131C14, 131C18, 130D25, 130D67 and 130D53 light chain variable region amplification was also performed using the respective primers listed in Table 1. The resulting vectors, pKLG1/131C14, pKLG1/131C18, pKLG1/130D25, and pKLG1/130D67 were confirmed by restriction enzyme digestion and sequencing.
Production of recombinant human anti-B5R antibody from CHO cells: For production of recombinant antibody, individual antibody vectors containing anti-B5R or H3L antibody were nucleofected into host cell dhfr-defective strain of Chinese Hamster Ovary cell (CHO cells, ATCC #CRL-9096) and recombinant antibody was isolated from the supernatant of the transfected cells. Briefly, 2 μg DNA of purified DNA expression vector was linearized by a DNA restriction enzyme, Asci, and the DNA was nucleofected into 1×107 cells CHO cells using the nucleofector kit V (Cat. No. VCA-1003) and the Amaxa nucleofector (Amaxa Biosystems) following manufacturer's instructions. The transfected cells were seeded in 96-well culture plates in EX-CELL 325 PF CHO serum-free medium with glutamine (JRH Bioscience, Lenexa, Kans.), supplemented with penicillin/streptomycin (BioWhitaker), HT (Sigma), and Geneticin (Invitrogen, Corp.) for selecting CHO cells containing the DNA vector. After the selection of several stable transfectant lines, high human IgG producers were identified by ELISA, and used for production of recombinant antibody.
Example 2This example describes exemplary heavy and light chain variable region sequences of antibodies that bind to B5R.
Nucleotide sequence of cDNA of 131C12 heavy chain variable region (HV) (from initiation codon (ATG) to the end of variable region) is as follows (SEQ ID NO:1):
Nucleotide sequence of cDNA of 131C12 light chain variable region (LV) (from initiation codon (ATG) to the end of variable region) is as follows (SEQ ID NO:3):
Nucleotide sequence of cDNA of 131C14 heavy chain variable region (HV) (from initiation codon (ATG) to the end of variable region) is as follows (SEQ ID NO:5):
Nucleotide sequence of cDNA of 131C14 light chain variable region (LV) (from initiation codon (ATG) to the end of variable region) is as follows (SEQ ID NO:7):
Nucleotide sequence of cDNA of C18 kappa light chain variable region (LV) (from initiation codon (ATG) to the end of variable region) is as follows (SEQ ID NO:9):
Nucleotide sequence of cDNA of C18 heavy chain variable region (HV) (from initiation codon (ATG) to the end of variable region) is as follows (SEQ ID NO:11):
Amino acid sequence of cDNA of 131C12 heavy chain variable region (HV) (leader sequence (bold) and variable region) is as follows (SEQ ID NO:2):
Amino acid sequence of cDNA of 131C12 light chain variable region (LV) (leader sequence (bold) and variable region) is as follows (SEQ ID NO:4):
Amino acid sequence of cDNA of 131C14 heavy chain variable region (HV) (leader sequence (bold) and variable region) is as follows (SEQ ID NO:6):
Amino acid sequence of cDNA of 131C14 light chain variable region-A (LV) (leader sequence (bold) and variable region) is as follows (SEQ ID NO:8):
Amino acid sequence of C18 kappa light chain variable region (LV) (leader sequence (bold) and variable region) is as follows (SEQ ID NO:10):
Amino acid sequence of C18 heavy chain variable region (HV) (leader sequence (bold) and variable region) is as follows (SEQ ID NO:12):
This example describes exemplary heavy and light chain variable region sequences of antibodies that bind to H3L.
Analysis of 130D25, 130D67 and 130D53 heavy and light chains revealed that they had the same heavy and light chain sequences. Hence, only the heavy and light chain variable region sequences of 130D67 are shown.
Nucleotide sequence of cDNA of 130D67 heavy chain variable region (HV) (from initiation codon (ATG) to the end of variable region) is as follows (SEQ ID NO:13):
Nucleotide sequence of cDNA of 130D67K-Light chain variable region (LV) (from initiation codon is as follows (SEQ ID NO:15):
Amino acid sequence of cDNA of 130D67 heavy chain variable region (HV) (leader sequence (bold) and variable region) is as follows (SEQ ID NO:14):
Amino acid sequence of cDNA of 130D67 light chain variable region (LV) (leader sequence (bold) and variable region) is as follows (SEQ ID NO:16):
This example includes a description of CDRs for exemplary B5R and H3L antibodies. This example also includes ATCC deposit numbers for exemplary B5R and H3L antibodies.
Exemplary CDR sequences (CDR1, CDR2 and CDR3) for variable region heavy (VH) and variable region light (VL) of exemplary B5R and H3L antibodies are set forth in Table 2 below:
The CDR sequences of light and heavy chain in Table 2 were identified using the NCBI Ig BLAST tool (CDR1 and CDR2 for both heavy and light chain) using the Kabat rules, and the position of CDR3 (CDR-L3 and -H3) was determined by applying the following rules:
The Cys residues are the most conserved feature.
For CDR-L1, start approximately at residue 24, Residue before a Cys, Residue after a Trp. Typically, for example, Trp-Tyr-Gln, but also, Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu. Length 10 to 17 residues.
For CDR-L2, start 16 residues after the end of L1, residues before generally Ile-Tyr, but also, Val-Tyr, Ile-Lys, or Ile-Phe. Length typically 7 residues (except NEW (7FAB) which has a deletion in this region).
For CDR-L3, start 33 residues after end of L2 (except NEW (7FAB) which has the deletion at the end of CDR-L2), residue before a Cys, residues after Phe-Gly-XXX-Gly (SEQ ID NO:123). Length 7 to 11 residues. For CDR-H1, start approximately at residue 26 (4 after a Cys) [Chothia/AbM definition], Kabat definition starts 5 residues later. Residues before Cys-XXX-XXX-XXX (SEQ ID NO:124), residues after a Trp. Typically Trp-Val, but also, Trp-Ile, Trp-Ala. Length 10 to 12 residues [AbM definition], Chothia definition excludes the last 4 residues.
For CDR-H2, start 15 residues after the end of Kabat/AbM definition of CDR-H1, residues before typically Leu-Glu-Trp-Ile-Gly (SEQ ID NO:108), but there are variations, residues after Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. Length Kabat definition 16 to 19 residues; AbM (and recent Chothia) definition ends 7 residues earlier.
For CDR-H3, start 33 residues after end of CDR-H2 (2 after a Cys), residues before Cys-XXX-XXX (typically Cys-Ala-Arg), and residues after Trp-Gly-XXX-Gly (SEQ ID NO:109). Length 3 to 25 residues.
ATCC deposit numbers for exemplary B5R and H3L antibodies are set forth in Table 3 below:
This example describes functional and molecular studies to characterize H3L and B5R antibodies.
The panel of 12 human anti-H3 mAbs was characterized in vitro by assays to identify anti-H3 mAb for further examination and development. One functional test was virus neutralization in vitro. Molecular characterizations also assess relatedness of the clones, isotypes and affinities. Supernatants from uncloned anti-H3 hybridoma cultures were pre-screened for neutralization activity. After cloning, anti-H3 mAbs of known concentration were screened in a conventional in vitro neutralization assay (Davies et al., J Virol 79:11724 (2005), Crotty et al., J Immunol 171:4969 (2003), Newman et al., J Clin Microbial 41:3154 (2003), Frey et al., N Engl J Med 346:1275 (2002)). Neutralization potency of each mAb was determined by quantitative dose titrations, measuring the lowest mAb concentration able to inhibit VACV infection 50%, as measured by plaque assay (PRNT50). The mAbs were analyzed in a series of three independent studies. Human mAbs were compared against several controls. Serum from an unvaccinated human was used as a negative control. Serum from a vaccinated human was used as a positive control. Mouse anti-H3 mAbs have also been isolated, and the best of those mAbs #41 was used as an additional comparison.
The results of analysis of anti-H3 mAbs are shown in
Anti-H3 mAbs were or will be subjected to molecular characterization. First, all anti-H3 mAbs were isotyped for both heavy chain and light chain, and all clones are human IgG1 κ. Second, estimates of relative affinities are determined by endpoint dilution ELISA using recombinant H3 as the capture antigen. Anti-H3 mAbs with higher affinities will generally report better endpoint dilution titers. Third, IEF gels will determine if all nine anti-H3 mAbs are independent clones, as indicated by distinct isoelectric points. Fourth, cross-blocking studies will determine if there are different epitopes on H3 recognized by the panel of 12 mAbs, and which mAbs are specific for the same (or overlapping) epitopes. Mouse mAbs will also be used in the cross-blocking studies to obtain more complete complementation groups.
Candidates for in vivo studies are determined by identifying the clones with potent in vitro neutralization activity. Clones with non-overlapping epitopes (i.e. clones 1, 2, and 3 for in vivo testing not all being specific to one epitope) are analyzed as different epitopes may have different levels of exposure in vivo. Finally, since all H3L clones thus far are human IgG1, and human IgG1 is a complement-fixing isotype, selection based on isotype at this point is unnecessary.
The panel of 11 B5R mAbs were characterized in vitro by assays to identify the best anti-B5 mAbs for further examination and development. One functional assay was direct neutralization in vitro. A second was a complement dependent neutralization assay (described in Example 8).
Vaccinia EEV neutralization is desired for an anti-B5 mAb to be used as a human immunotherapeutic. However, neutralization of EEV is less well defined than neutralization of IMV. EEV particles are highly labile and therefore it is difficult to produce EEV stocks (Smith et al., J Gen Virol 83:2915 (2002), Amanna et al., Immunol Rev 211:320 (2006), Isaacs et al., J Virol 66:7217 (1992), Lustig et al., Virology 328:30 (2004)). Furthermore, direct neutralization of EEV is very inefficient, with ˜50% neutralization as a common best case scenario (Aldaz-Carroll et al., J Virol 79:6260 (2005)). One assay that resolves some of these problem is the comet tail inhibition assay (Amanna et al., Immunol Rev 211:320 (2006), Galmiche et al., Virology 254:71 (1999)).
Some poxvirus strains, particularly if left for a longer time (e.g. 72 hrs) will form “comet tail” plaques. The comet tails are due to the release of EEV virions from infected cells, and the EEV particles diffuse a short distance before infecting new cells. The directionality of the comet tail is due to weak convection currents in the medium. Comet tail formation can be inhibited by addition of anti-B5 antibodies to medium (Lustig et al., J Virol 79:13454 (2005), Bell et al., Virology 325:425 (2004), Lustig et al., Virology 328:30 (2004), Viner et al., Microbes Infect 7:579 (2005)).
The full panel of human anti-B5 mAbs were analyzed by comet tail inhibition. VACVIHDJ was used in the assay as it produces a higher percentage of EEV than VACVNYBOH and thereby more distinct comet tails. VACVIHDJ is used by other research groups studying EEV for this reason.
Hela cells were infected at low density (40 PFU in a 6 well dish) and then overlaid with medium containing 20 μg/ml anti-B5 mAb. Serum from a vaccinated donor with the highest anti-B5 titer was used as a positive control (at a 1:10 dilution), and serum samples from several unvaccinated donors were used as negative controls (also at 1:10 dilution). Cells were incubated for 72 hrs without disturbance and then developed with crystal violet fixation to observe comet tails. Positive control serum from a vaccinated donor with the highest anti-B5 IgG titer gave nearly complete inhibition of comet tail formation (
While the comet tail in vitro assay can measure functional anti-EEV neutralization, this assay has two limitations. First, comet tails can be inhibited by anti-EEV B5-specific antibodies, but the inhibition requires a high concentration of antibody-20-40 μg/ml is frequently used. This is ˜500× more IgG than the amount of anti-H3 mAb needed to neutralize IMV. It is also much higher than the IgG concentrations required to neutralize a wide variety of viruses in vitro, such as influenza, SARS, polio, and rabies. Such high levels of anti-B5 IgG may not be physiological. Second, anti-EEV antibodies targeting the A33 protein are protective in vivo but do not inhibit comet tail formation, indicating a distinction between in vitro EEV comet tail inhibition and in vivo EEV neutralization (Lustig et al., J Viral 79:13454 (2005), Galmiche et al., Virology 254:71 (1999)). Given these limitations, the power of the comet tail assay to predict which anti-B5 mAbs would be most efficacious in vivo is limited. Thus, the complement dependent neutralization assay described in Example 8 can be used as a primary endpoint.
B5R mAbs were also subjected to the molecular characterizations described above for H3 mAbs. The 11 human anti-B5 mAbs were isotyped for both heavy chain and light chain (All κ). Six of 11 were IgG1, three were IgG4, one was IgG3, and one was IgG2. Second, estimates of relative affinity of the purified clones are determined by endpoint dilution ELISA using recombinant B5 as the capture antigen in a standard BIAcore assay using purified mAbs. MAbs with higher affinities will generally report better endpoint dilution titers. Third, IEF gels were run to determine if all six of the most promising mAbs are independent clones, as indicated by distinct isoelectric points.
Cross-blocking studies were performed to determine the number of different epitopes on B5 recognized by a panel of mAbs, and which mAbs are specific for the same (or overlapping) epitopes. An ELISA was used to determine if the antibodies bind to the same B5R epitope.
In brief, NUNC 96 well flat bottom ELISA plates were coated with individual mouse or human anti-B5R antibodies in carbonate buffer at 2 μg/ml for 1 hour at 37° C. The plates were washed and then blocked with PBS/1% BSA/Tween 20. Anti-B5R antibodies were then pre-incubated with recombinant vaccinia virus 6×His tagged soluble B5R for 30 minutes at 4° C. The combinations of antibody-protein were added to the plate and incubated for 1 hour at 4° C. After 3 washes, bound B5R-6×His was detected with peroxidase conjugated anti-6×His epitope tag Ig (Clonetech). C12 and C14 are human anti-B5R antibodies and B96, B116 and B126 are mouse anti-B5R antibodies. The percent inhibition was determined using the OD of each sample in the following formula: % inhibition=(max−sample/max)*100. Max represents the OD from the sample with no inhibiting Ab or hIgG isotype control. The cross-blocking results are illustrated in Tables 4A and 4B:
The data show that C12, C14, C18, C33 and C30 block each other and are therefore members of the same epitope group. B116, B126 and 896 are not blocked, and therefore each represent distinct epitope groups. In toto, there are five epitope groups (Groups A-D) recognized by the anti-B5R antibodies based on competition for binding to recombinant B5R protein (B5R-6×His) by ELISA. Top B5R antibody candidates bind to the overlapping epitope groups: C12 and C14 seem to recognize the same or very closely related epitope, and C18 seem to recognize an epitope cluster overlapping with but not identical to that of C12 and C14.
Example 6This example describes in vivo efficacy studies of B5R and H3L antibodies, using three standard lethal challenge vaccinia models.
There are three in vivo models used to study the ability of mAb pretreatment to protect mice. The first is intranasal challenge of BALBc with VACVWR. The second is intravenous challenge of SCID mice with VACVWR vaccine strain. The third is intravenous challenge of SCID mice with VACVNYBOH vaccine strain. Studies with these models are described in detail below.
All 10 anti-B5 mAbs were analyzed for the ability to protect BALBc mice from a lethal intranasal challenge with VACVWR. This is the same assay used in the studies described in Example 4 and is widely used as a model for smallpox inhalation in humans (Ramirez et al., J Gen Virol 83:1059 (2002), Zhang et al., J Virol 74:11654 (2000), Belyakov et al., Proc Natl Acad Sci USA 100:9458 (2003), Alcami et al., Cell 71:153 (1992)). In this regard, the respiratory route of infection of VACVWR and smallpox are the same. An excellent feature of this model is that while the primary endpoint is survival, weight loss can also be tracked as a quantitative measure of protection. Both severity (nadir) and duration of weight loss can be used as criteria to measure the severity of the infection in instances of partial protection.
In brief, each mouse was administered 100 μg of a particular anti-B5 mAb i.p. at day −1 (5 mice/group). After light anesthesia (isofluorane), mice were infected intranasally with 3×104 PFU VACVWR (2 LD50) in a 10 μl volume. Mouse weight was measured daily for a period of four weeks. Any mouse with 30% weight loss was euthanized as per the animal protocol, based on earlier work showing mice do not recover from that severe weight loss. Mice were also examined for clinical symptoms (lethargy, ruffled fur, hunched back) that can be composited into a mean clinical disease score (similar to Crotty et al., Blood 108:3085 (2006)). The human mAbs were compared to a murine mAb #B96.
In a first study, human anti-B5 mAbs #C14 and #C33 provided 100% protection from death and provided better protection against weight loss than the mouse mAb #B96 (
Using similar conditions, the protective efficacy of several human anti-B5 mAbs were studied in SCID mice infected with VACVWR, a more virulent VACV strain than VACVNYBOH. The data is shown in
In brief, groups of 5 SCID mice were administered VIG, 100 μg anti-B5 human mAb #C12, 100 μg anti-B5 human mAb #C14, or PBS at day −1, then infected i.v. with 1×103 PFU VACVWR on day 0. Mice pretreated with either human anti-B5 mAb (C12, C14) were well protected from VACVWR. The anti-B5 mAb treated mice had minimal weight loss and were all alive at day 14, while untreated mice (PBS) rapidly lost weight and all died by day 14. The anti-B5 mAbs both protected significantly better than VIG (
The full panel of anti-H3 human mAbs, which have been characterized for virus neutralization in vitro, are tested in vivo. The best three candidates based upon neutralization assays are #67, #25, and #53, with #78 and #58 also being of similar potency. Antibody #54 is a suitable negative control mAb, since it exhibited no detectable neutralization in vitro. The VACVWR BALBc i.n. challenge system and the VACVNYBOH SCID i.v. challenge system are used as described above for anti-B5. Since anti-H3 antibodies are protective in vivo (Davies et al., J Virol 79:11724 (2005)), rabbit anti-H3 serum is used as a positive control.
An in vivo protection study with mouse anti-H3 mAb #41 was performed. In brief, SCID mice were administered VIG, 200 μg anti-H3 #41 or PBS at day −1, then infected i.v. with 1×103 PFU VACVWR on day 0. The mice administered anti-H3 #41 exhibited substantial protection from weight loss for greater than two weeks after infection with virulent VACVWR (
Combination anti-H3L and anti-B5R antibody therapy is therefore predicted to be very potent, which is also demonstrated below.
The third in vivo assay used as a screen for candidate mAbs is the animal model considered to resemble closely human progressive vaccinia. In this system, SCID mice are infected with smallpox vaccine VACVNYBOH and then measured for weight loss, pox formation, and death. This model has been used by VIG vendors (Shearer et al., Antimicrob Agents Chemother 49:2634 (2005)) and the FDA (Goldsmith et al., Vox Sang 86:125 (2004)). This model is valuable both because it uses the human vaccine strain of vaccinia and immunodeficient or immunocompromised humans are a group of concern for failing to control VACV and develop progressive vaccinia or other life threatening side effects, which are the clinical conditions for which VIG is licensed.
In this model, treatment with a human mg/kg equivalent dose of VIG is able to extend life by >1 week (Shearer et al., Antimicrob Agents Chemother 49:2634 (2005)), (Goldsmith et al., Vox Sang 86:125 (2004)). Since SCID mice are fully immunodeficient, a complete cure is not expected, but life extension correlates with the ability of VIG to reduce or prevent severe vaccinia side effects in humans, and an effective post-exposure prophylactic against smallpox. Since this assay uses the human smallpox vaccine strain, models vaccinia infection in an immunodeficient/immunocompromised setting, and has been used at the FDA, this animal model is useful for identifying successful candidate mAbs.
In brief, groups of six SCID mice were administered anti-H3 mAb #41+human anti-B5 mAb C14 (200 μg and 100 μg respectively) (
These mice were also scored for percent survival, development of pox lesions and those mice that remained disease free (
Work by other groups has shown that after initiation of infection, VIG can have a modest partial effect given post-exposure at day +2 to +4 (Shearer et al., Antimicrob Agents Chemother 49:2634 (2005)). Given the data with the more virulent VACVWR strain, human mAbs will likely outperform VIG.
The mAbs were compared to the commercially available “standard of care” VIG therapy. Mabs are considered “noninferior” to VIG if they extend survival time by an equivalent number of days, and mAbs are considered “superior” to VIG if they extend survival time longer than VIG, as measured by standard Kaplan-Meier survival curve statistical analysis.
In brief, groups of six SCID mice were given anti-H3 mAb #41 (200 μg)+human anti-B5 mAb C14 (100 μg) (
These mice were also scored for percent survival, development of pox lesions and for those that remained disease free (
WorkReports by others groups has have shown that after initiation of infection, VIG can have a modest partial effect given post-exposure at day +2 to +4 (Shearer et al., Antimicrob Agents Chemother 49:2634 (2005)). Given the data with the more virulent VACVWR strain, human mAbs antibodies will likely outperform VIG.
Example 7This example includes a description of additional assays useful in characterizing function and broad reactivity spectrum of antibodies.
Demonstration of immunological cross-reactivity of top antibodies with variola virus homologs: H3 protein is highly conserved among orthopox viruses. The H3 protein sequence is 96-98% conserved between VACV and variola (smallpox), and 94% conserved between VACV and monkeypox. B5 has similarly high levels of conservation, close to 92%. For some mAbs, Western blot analysis can be used to check their cross-reactivity in vitro.
Bacterially expressed recombinant full-length B6 protein and its fragments: 825 nucleotides of the predicted nucleotide sequence coding for amino acids 1-275 of the variola virus protein B6 (accession # X65519) were synthesized by GenScript Corporation (Piscataway, N.J., USA). Five vectors for the expression of various lengths of the B6 protein were produced using bacterial expression vector pMAL-C4X (New England BioLabs, Ipswich, Mass., USA). The pMAL-C4X vector contains the malE gene for expression of maltose-binding protein (MBP). All of the vectors were constructed such that the nucleotides coding for the B6 protein were inserted downstream of, and in the same translational reading frame as, the malE gene. As a result, the translation initiating with the MBP protein should continue through the coding region of the B6 forming an MBP-B6 fusion protein. In general, the resulting fusion proteins consist of 391 amino acids at the amino-terminal end coding for the maltose-binding protein followed by the portion of B6 protein coded for by the region of B6 cloned into that particular expression vector. Constructs encoding B6 fragments were designed to contain overlapping short consensus repeat sequences (SCR) to facilitate narrowing down epitope specificity for top anti-B5 mAbs. The five expression vectors created are: 1) pMAL-B6R (B6 amino acids 20-275), 2) pMAL-B6R(SCR1-SCR2) (B6 amino acids 20-132), 3) pMAL-B6R(SCR2-SCR3) (B6 amino acids 71-184), 4) pMAL-B6R(SCR3-SCR4) (B6 amino acids 133-275), 5) pMAL-B6R (20-100) (B6 amino acids 20-100). All of the above vectors were constructed using the same technique. Briefly, oligonucleotide primers specific for the regions of interest were designed containing 5′-EcoRI and 3′-HinDIII restriction endonuclease recognition sites: B6 (20-275) (primers SCR1_EcoR1_F58, ExCell_HindIII_R828), B6 (20-132) (primers SCR1_EcoR1_F58, SCR2_HindIII_R396), B6 (71-184) (primers SCR2_EcoR1_F211, SCR3_HindIII_R552), B6 (133-275) (primers SCR3_EcoR1_F397, ExCell_HindIII_R828), B6 (20-100) (primers SCR1_EcoR1_F58, B6R_HindIII_R300). (Table 6) Respective oligonucleotide primers were used to amplify B6 gene fragments from the B6 template by polymerase chain reaction (PCR) with KOD Hot Start DNA polymerase (EMD Chemicals, Inc., Gibbstown, N.J., USA). The resulting nucleotide fragments were digested with restriction enzymes EcoRI and HinDIII (New England BioLabs), as was the pMAL-C4X vector. All nucleotide fragments were run on agarose gels, and DNA bands corresponding to the theoretical nucleotide size were extracted and purified by column purification (QIAquick Gel Extraction Kit, Qiagen Inc., Valencia, Calif., USA). Each of the B6 nucleotide fragments were separately ligated into the pMAL-C4X vector using DNA Ligase Mighty Mix (Takara Bio USA, Madison, Wis., USA). Ligation mix was transformed into E. coli bacteria strain DH5a (Invitrogen Corp., Carlsbad, Calif., USA) and grown on Luria-Bertani (LB) broth (MP Biomedicals, LLC, Solon, Ohio, USA) agarose plates containing ampicillin (100 ug/mL) (Sigma-Aldrich, St. Lois, Mo., USA). Colonies were grown in LB broth with ampicillin, plasmid DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen, Inc.), analyzed by restriction digest and sequenced to verify nucleotide sequence of the B6 fragments (GENEWIZ, Inc., San Diego, Calif., USA).
B6 Bacterial Expression:
B6 expression vectors were transformed into E. coli bacteria strain DH5a and grown on LB broth-agarose plates containing ampicillin (100 ug/mL). Individual colonies were grown over night (˜18 hours) at 37° c. shaking at 250 rpm in liquid LB containing ampicillin (100 ug/mL). Cultures were diluted 1 part in 20 into two containers of fresh LB with ampicillin and incubated as before. When cultures reached an optical density at a wavelength of 600 nm (OD600) of 0.5-0.8, one of the duplicate cultures was induced to express the B6 protein by adding isopropyl •-D-1-thiogalactopyranoside (IPTG) (BioPioneer, Inc., San Diego, Calif., USA) to a final concentration of 1 mM; the other paired culture was not induced. The cultures were grown as before for three hours. Equal volumes of each culture were removed, bacteria pelleted by centrifugation, supernatant removed, and pellets were lysed by adding 2× Laemmli buffer (Laemmli UK, Nature 227, 680-685, 1970) and incubating at 95° C. for 5 minutes.
B6 Sequences
Nucleotide sequence of the extracellular domain of variola virus B6 cDNA (accession # X65519) from initiation codon (ATG) to the end of extracellular domain (nucleotides 1-825) SEQ ID NO:117
Amino acid sequence of Variola major virus, strain India-1967, B6 extracellular domain (amino acids 1-275, leader sequence (bold)); SEQ ID NO:118
Demonstration of cross-reactivity of the lead anti-B5R mAbs with variola virus homolog by Western blot:
All top anti-B5R mAbs demonsrated their reactivity against B5R in Western blot conditions. Based on this result, Western blot assay could be used to check their cross-reactivity with variola homolog, B6. In this assay, adequate amounts of B6 ectodomain, its fragments, and B5R protein were blotted on PVDF membrane (0.45 um, Invitrogen) following electrophoresis on SDS PAGE (4-20% Tris-Glycine gels, Invitrogen). The blocked with milk membranes were incubated with 131C12, 131C14, or 131C18 human mAbs, and their binding was detected with goat anti-human IgG Fcγ HRP conjugate (Jackson Immunoresearch Labs). The antigen bands were visualized on X-ray film following exposure of the membranes pre-incubated with h ECL Plus™ chemillimunescence substrate (GE Healthcare).
Based on the western blot data, all top human anti-B5R mAbs demonstrated strong cross-reactivity with variola homolog protein, B6 (
Narrowing down epitope specificity for the top human anti-B5R mAbs by Western blot: The epitope specificity of human anti-B5R mAbs was narrowed down to N-terminal portion of B5R protein, including SCR1, based upon the demonstrated reactivity towards fragments of B6 protein (
Cloning of vaccinia and variola H3L ectodomains: Homologous nucleotide regions coding for amino-terminal extracellular domains of the H3 protein from variola virus strain Lister (VACVlis) (accession # AY678276), and variola major virus strain Bangladesh-1975 (VARVban) (accession # L22579) were separately cloned into bacterial expression vector pET21a(+) (EMD, Novagen Brand, Madison, Wis., USA) such that translation of the H3 coding frame would add a six histidine tag (6-HIS tag), coded for by nucleotides in the vector, at the carboxy-terminus of the H3 protein. Specifically, 807 nucleotides of the predicted nucleotide sequence coding for amino acids 1-269 of the H3 protein from VACVlis, and 810 nucleotide of the predicted nucleotide sequence coding for amino acids 1-270 of the H3 protein from VARVban were synthesized by GenScript Corporation (Piscataway, N.J., USA). Nucleotides coding for the restriction endonuclease sites of NdeI and XhoI were added at the 5′ and 3′ ends respectively of each H3 nucleotide fragment during synthesis. The nucleotide fragments were digested with restriction enzymes NdeI and XhoI, as was vector pET21a(+). Resulting H3 and pET21a(+) nucleotide fragments were isolated by agarose gel electrophoresis and purified using QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, Calif., USA). Purified H3 nucleotide fragments were then ligated individually into the digested pET21a(+) vector, transformed into E. coli bacteria strain DH5α (Invitrogen Corp., Carlsbad, Calif., USA) and grown on Luria-Bertani (LB) broth (MP Biomedicals, LLC, Solon, Ohio, USA) agarose plates containing ampicillin (100 ug/mL) (Sigma-Aldrich, St. Lois, Mo., USA). Colonies were grown in LB broth with ampicillin, plasmid DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen, Inc.), analyzed by restriction digest and sequenced to verify nucleotide sequence of the H3 fragments (GENEWIZ, Inc., San Diego, Calif., USA).
Protein Expression: Individually, H3 expression vectors were transformed into E. coli bacteria strain DH5α and grown on LB broth-agarose plates containing ampicillin (100 ug/mL). Individual colonies were grown over night (˜18 hours) at 37° c. shaking at 250 rpm in liquid LB broth containing ampicillin (100 ug/mL). Cultures were diluted 1 part in 20 into two containers of fresh LB with ampicillin and incubated as before. When cultures reached an optical density at a wavelength of 600 nm (OD600) of 0.5-0.8, one of the duplicate cultures was induced to express the H3 protein by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) (BioPioneer, Inc., San Diego, Calif., USA) to a final concentration of 1 mM; the other paired culture was not induced. The cultures were grown as before for three hours. Equal volumes of each culture were removed, bacteria pelleted by centrifugation, supernatant removed, and pellets were lysed by adding 2× Laemmli buffer (Laemmli UK, Nature 227, 680-685, 1970) and incubating at 95° C. for 5 minutes.
H3L Ectodomains Nucleotide Sequences
Synthesized nucleotide sequence of H3 from vaccinia virus strain Lister (from NdeI restriction site (CATATG) through XhoI restriction site (CTCGAG). Start codon is ATG.) SEQ ID NO: 119
Synthesized nucleotide sequence of H3 from variola major virus strain Bangladesh-1975 (from NdeI restriction site (CATATG) through XhoI restriction site (CTCGAG). Start codon is ATG.) SEQ ID NO:120
Amino acid sequence of H3 protein from vaccinia virus strain Lister expressed from vector pET21a(+) (amino acids 1-269 of H3 through the 6-HIS tag coded by the vector (underlined)) SEQ ID NO:121
Amino acid sequence of H3 protein from variola major virus strain Bangladesh-1975 expressed from vector pET21a(+) (amino acids 1-269 of H3 through the 6-HIS tag coded by the vector (underlined)) SEQ ED NO 122:
Demonstration of cross-reactivity of human anti-vaccinia H3L 130D67 mAb with variola virus homolog by Western blot:
Western blot assay confirmed that 130D67 human mAb raised against vaccinia H3L cross-reacts with variola homolog. The antibody showed strong recognition of H3L ectodomain both from vaccinia and variola viruses (
Neutralization of variola and monkeypox: While VIG lots are not directly tested for efficacy against variola or monkeypox, mAbs can be assayed for neutralization of variola and/or monkeypox. Given that the best available data indicates that post-exposure treatment with VIG is ˜75% effective against smallpox (Hopkins et al., Clin Infect Dis 39:819 (2004)), a national or military VIG stockpile would likely be dual purpose-treatment of smallpox vaccine side effects, and emergency treatment of a smallpox outbreak.
Ng/NDA VACVWR eczema vaccinatum model: A mouse model for eczema vaccinatum has been established (Kawakami et al., Alergol. Int. Epub, 56, September (2007)) Given that eczema vaccinatum is a concern of the smallpox vaccine, the ability of mAb therapy to treat eczema vaccinatum is studied. Atopic dermatitis is induced in Ng/NDA mice, which are then infected with VACVWR at the site of the dermatitis. Eczema vaccinatum is measured by lesion size and clinical score. Mice are treated with anti-B5 mAb, anti-H3 mAb, or VIG intravenously immediately prior to VACVWR scarification. Protection is measured by reduction of eczema vaccinatum lesion size and lesion duration. In a separate set of studies, protection is measured by reduction of viral loads in the lesion (at the site of infection) at day 7, and prevention/reduction of viral spread as measured by viral titers in lung at day 7 post-infection. Skin biopsies can also be taken from this study at day 7, and pathology scoring of histological sections from each animal will be done to measure severity of epithelial damage and leukocyte infiltration.
SCID VACVWR tail pocks model: This is a model that has been used by investigators to query protection against pock/lesion formation after VACV infection. While the lesions are not histologically the same as human smallpox, monkeypox, or vaccinia infection, the model does recapitulate the basic phenomenon of distinct viral skin pustules. While this assay has been around for 40 years (Joshi et al., Appl Microbiol 18:935 (1969)), there can be large variability between individual mice, making it necessary to use large numbers of animals to determine statistically significant differences (Shearer et al., Antimicrob Agents Chemother 49:2634 (2005)). Groups of 12 BALBc mice are treated with mAbs or VIG at day −1 and then infected with 2×105 PFU VACVNYBOH at day 0 by subcutaneous injection of the tail. Necrotic pocks on the tail skin are visualized at day 8 by counterstaining (Shearer et al., Antimicrob Agents Chemother 49:2634 (2005)), Joshi et al., Appl Microbial 18:935 (1969)) and quantified. If VACVNYBOH vaccine strain fails to give sufficient pocks, a more virulent VACV strain (VACVWR or VACVIHD-J) can be used, as has been done by other investigators (CangeneCorporation, (ed. WIPO) (2003), Neyts et al., Antimicrob Agents Chemother 46:2842 (2002), Spriggs et al., Proc Natl Acad Sci USA 89:6070 (1992)).
Example 8This example includes a description of a new EEV neutralization assay that correlates with in vivo protection against vaccinia. This example also includes in vivo protection data and data generated with this EEV neutralization assay.
Conventional neutralization assays are done in the absence of complement (using heat inactivated serum) and are what can be called “direct neutralization” assays. While such assays provide useful information, it is likely that aspects of neutralization in vivo are heavily influenced by complement and mAbs are likely to vary in their virus neutralization in the presence of complement (see, for example, B5, described later). Thus, it is useful to assay virus neutralization in the presence of complement. Given that the EEV comet tail inhibition assay is limited in terms of predicting protective efficacy of anti-B5 mAbs in vivo (Table 5), and EEV are resistant to conventional direct neutralization, a new in vitro EEV neutralization assay that more accurately predicts or correlates with in vivo protection was developed. This assay can be used to develop data to provide greater in vivo relevancy for comparing functional antiviral characteristics of mAbs vs. VIG.
In brief, VACVWR (80 PFU) EEV (standard preparation of secreted EEV as the 48 hr supernatant of VACVWR infected HeLa cells, titered and kept at 4° C. for ≦30 days, ref. (Bell et al., Virology 325:425 (2004), Lustig et al., Virology 328:30 (2004), Viner et al., Microbes Infect 7:579 (2005)) was incubated with 10% rabbit complement plus 1 μg/ml anti-B5 mAb for 60 min at 37° C., then added to Vero cells. After 1 hr, virus was washed from the monolayer and cells were incubated 42 hrs at 37° C. and then developed with crystal violet and VACV plaques were enumerated. This assay was performed on a large panel of human and murine anti-B5 mAbs (
In brief, this was confirmed in vivo: mice were administered 100 μg of a particular anti-B5 mAb i.p. (or PBS as a negative control) at day −1 (5 mice/group). After light anesthesia (isofluorane), mice were infected intranasally with 3×104 PFU VACVWR (2 LD50) in a 10 μl volume. Mouse weight was measured daily. Any mouse with 30% weight loss was euthanized. Monoclonal antibody B126 was highly protective, while B96 was weakly protective (
The data demonstrate a high correlation between in vitro neutralization activity in the presence of complement and protection in vivo against a lethal VACVWR challenge (
The best comet tail inhibiting mAB, #B96, did not exhibit complement-mediated EEV neutralization, nor was this antibody effective in vivo (
This example includes demonstration of the significant role of the complement in the protection mechanism of anti-B5 antibodies.
Requirement for complement in vivo. B126 antibody was distinguished from other murine anti-B5 mAbs by its ability to neutralize EV in vitro. The neutralization assay incorporated complement. B126 was also the only murine IgG2a clone identified, and IgG2a is the most efficient complement binding murine isotype. These findings indicated that the potent protective efficacy of B126 in vivo was likely due to its ability to fix complement. To confirm this hypothesis the efficacy of B126 after depleting complement was analyzed in vivo. Complement was depleted by administering cobra venom factor (CVF), which was confirmed by measuring serum levels of C3 (94% depletion,
The physiological EV neutralization assay in vitro demonstrated that anti-B5 antibodies can efficiently neutralize EV virions, and this functionality was predictive of protective efficacy in vivo. Complement can function in multiple ways, and the observation that complement fixing mAbs were so effective in vivo that mice developed no clinical symptoms suggested that the VACV infection may be stopped very rapidly. These observations mean that anti-B5 antibodies are likely able to direct complement-mediated destruction of VACV infected cells, utilizing the membrane attack complex, and thereby rapidly quenching the spread of VACV, leading to resolution of the infection.
The ability of anti-B5 mAbs to direct complement lysis of cells infected with VACV was studied. While MV VACV virions (MV), the most abundant virion form, are produced intracellularly and do not have cell surface exposed proteins, EV are secreted from the plasma membrane. VACV infected cells express B5 on the surface. This expression can be detected within 4 hours of infection, and is at high levels by 8-10 hours (infected MFI 500 vs. uninfected MFI 16,
Adherent cells were infected with VACV and examined for susceptibility to antibody directed complement lysis at time points after infection when virus expression of B5 protein led to an accumulation of B5 on the surface of infected cells (8-12 hrs). Treatment with antibody or complement alone had no effect on infected cells (
Similar data on in vitro direct complement-mediated lysis of VACV infected cells were obtained with human antibodies (
Antibody modifications leading to increased Fey complement fixation have been described (see, for example, Idusogie E E, Wong P Y, Presta L G, et al. Engineered antibodies with increased activity to recruit complement. J Immunol 2001; 166:2571-5). Several studies have reported that CDC can be enhanced through improved C1q binding as a result of engineered Fc or hinge antibody regions (Dall'Acqua et al. J Immuno; 177:1129 (2006); Michaelsen et al. Scand J Immuno; 32:517 (1990)8; Brekke et al. Mol Immunol 30: 1419 (1993) Scientists of Kyowa-Hacco Kogyo Co., Ltd. have described an approach to generate highly potent CDC activity by creating unique chimeric Fcγ construct sharing domains of human IgG1 and IgG3 isotypes (see, Natsume et al. Cancer Res 68:3863 (2008). The data indicate that anti-B5 antibodies with modifications enhancing complement fixation and CDC will have enhanced protective potency in vivo and therefore greater therapeutic value.
Example 10This example includes a description of in vivo protection data of human anti-B5 antibody, anti-H3 antibody, and a combination therapy with anti-B5 antibody and anti-H3 antibody. A combination of two mAbs specific to H3L and B5R were compared to individual mAbs and VIG for in vivo protection of mice, as described in Example 6. This study was done with fully human mAbs.
In brief, SCID mice were injected at Day −1 with 100 μg human anti-B5 (C14), 200 μg human anti-H3 (#67), a combination of both, or VIG (1.25 mg per mouse). Mice were infected at Day 0 with VACVNYBOH i.v. 1×104 PFU.
Individual administration of human mAbs specific for H3L or B5R provided equivalent protection to VIG, measured by weight loss (
This example includes a description of in vivo protection data of combination therapy with anti-B5 antibody and VIG.
Using an intranasal VACVWR challenge model, fully human mAbs can be used to supplement VIG. In brief, mice were injected at Day −1 with 600 μg human VIG, or 600 μg human VIG supplemented with 0.5 μg human anti-B5 mAb (C12), or 600 μg human VIG supplemented with 5.0 μg human anti-B5 mAb (C12), or PBS. Mice were infected at Day 0 with VACVwr i.n. (5×104 PFU).
Mice treated with VIG supplemented with 0.5 or 5.0 μg human anti-B5 mAb (C12) exhibited better heath than mice provided VIG alone (
Supplementing VIG with anti-B5 human monoclonal antibody (Mab) had a significant benefit protecting mice. The data therefore indicate that monoclonal antibodies can be used to supplement VIG and provide therapeutic enhancement.
Claims
1. An isolated or purified antibody or subsequence thereof that specifically binds to poxvirus B5R envelope protein, wherein said antibody or subsequence comprises a heavy chain variable region sequence comprising CDR1, CDR2 and CDR3 sequences set forth as SSAMS, VISISGGSTYYADSVKG, and ETRYYYSYGMDV (SEQ ID NOs:17-19), respectively, and a light chain variable region sequence comprising CDR1, CDR2 and CDR3 sequences set forth as RASQRIGFALA, DASSLET, and QQFNTYPFT, (SEQ ID NOs:29-31) respectively.
2. An isolated or purified antibody or subsequence thereof that specifically binds to poxvirus H3L envelope protein, wherein said antibody or subsequence comprises a heavy chain variable region sequence comprising CDR1, CDR2 and CDR3 sequences set forth as DYAIH, GISWNGRSIGYADSVKG, and DIGFYGSGSLDY (SEQ ID NOs:26-28), respectively and a light chain variable region sequence comprising CDR1, CDR2 and CDR3 sequences set forth as RASQSVSSYLA, DASNRAT and QQRSNWPALT (SEQ ID NOs:38-40), respectively.
3. An isolated or purified antibody or subsequence thereof that specifically binds to poxvirus B5R envelope protein, wherein said antibody or subsequence comprises a heavy chain variable region sequence comprising CDR1, CDR2 and CDR3 sequences set forth as SYSMN, SISSSRSFIYYADSVKG and ERRYYYSYGLDV (SEQ ID NOs:20-22), respectively, and a light chain variable region sequence comprising CDR1, CDR2 and CDR3 sequences set forth as RASQGISSALA, DASSLES and QQFNSYPYT (SEQ ID NOs:32-34) respectively.
4. An isolated or purified antibody or subsequence thereof that specifically binds to poxvirus B5R envelope protein, wherein said antibody or subsequence comprises a heavy chain variable region sequence comprising CDR1, CDR2 and CDR3 sequences set forth as SYSMN, SISSSSSYIYYADSVKG and ERRYYYSYGMDV (SEQ ID NOs:23-25), respectively, and a light chain variable region sequence comprising CDR1, CDR2 and CDR3 sequences set forth as RASQRIGFALA, DASSLET and QQFNTYPFT (SEQ ID NOs:29-31), respectively.
5. The antibody of claim 1, 3 or 4, wherein the antibody inhibits at least 50% of the binding of the antibody or subsequence thereof of claim 1 or 3 or 4 to variola poxvirus B5R envelope protein or to poxvirus B6 envelope protein, or to the monkeypox B5 homolog, as determined in an ELISA assay.
6. The antibody of claim 2, wherein the antibody inhibits at least 50% of the binding of the antibody or subsequence thereof of claim 2 to poxvirus H3L envelope protein, as determined in an ELISA assay.
7. The antibody subsequence of any of claims 1 to 4, wherein the antibody subsequence is selected from Fab, Fab′, F(ab′)2, Fd, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv) and VL and VH domain fragments.
8. The antibody or the subsequence thereof of any of claims 1 to 4, wherein the antibody is produced by a hybridoma deposited as PTA-8654; PTA-8562; PTA-8653; or PTA-8564 (ATCC 110801 University Blvd., Manassas, Va. 20110-2209).
9. A nucleic acid that encodes the antibody or the subsequence of any of claims 1 to 4.
10. A vector comprising a nucleic acid that encodes the antibody or the subsequence of any of claims 1 to 4.
11. A host cell that expresses the antibody or the subsequence of any of claims 1 to 4.
12. A pharmaceutical composition, comprising the antibody of any of claims 1 to 4, and a pharmaceutically acceptable excipient or carrier.
13. A method for providing a subject with protection against poxvirus infection or pathogenesis, comprising administering an amount of the antibody or subsequence thereof of any of claims 1 to 4 sufficient to provide the subject with protection against poxvirus infection or pathogenesis.
14. A method for protecting or decreasing susceptibility of a subject to a poxvirus infection or pathogenesis, comprising administering an amount of the antibody or subsequence thereof of any of claims 1 to 4 sufficient to protect or decrease susceptibility of the subject to poxvirus infection or pathogenesis.
15. A method for decreasing or preventing an adverse side effect or complication associated with or caused by vaccination with a Vaccinia virus, comprising administering an amount of the antibody or subsequence thereof of any of claims 1 to 4 to a subject sufficient to decrease or prevent an adverse side effect or complication associated with or caused by vaccination with a Vaccinia virus.
16. The method of claim 13, further comprising administering an additional antibody or subsequence thereof that binds to a poxvirus protein.
17. The method of claim 16, wherein the additional antibody comprises VIG.
18. The isolated or purified antibody or subsequence thereof of claim 1, wherein said antibody or subsequence comprises a heavy chain variable region sequence set forth as SEQ ID NO:2 or a light chain variable region sequence set forth as SEQ ID NO:4.
19. The isolated or purified antibody or subsequence thereof of claim 2, wherein said antibody or subsequence comprises a heavy chain variable region sequence set forth as SEQ ID NO:14 or a light chain variable region sequence set forth as SEQ ID NO:16.
20. The isolated or purified antibody or subsequence thereof of claim 3, wherein said antibody or subsequence comprises a heavy chain variable region sequence set forth as SEQ ID NO:6 or a light chain variable region sequence set forth as SEQ ID NO:8.
21. The isolated or purified antibody or subsequence thereof of claim 4, wherein said antibody or subsequence comprises a heavy chain variable region sequence set forth as SEQ ID NO:12 or a light chain variable region sequence set forth as SEQ ID NO:10.
22. The method of claim 14, further comprising administering an additional antibody or subsequence thereof that binds to a poxvirus protein.
23. The method of claim 22, wherein the additional antibody comprises VIG.
24. The method of claim 15, further comprising administering an additional antibody or subsequence thereof that binds to a poxvirus protein.
25. The method of claim 24, wherein the additional antibody comprises VIG.
| 7393533 | July 1, 2008 | Crotty et al. |
| 03/068151 | August 2003 | WO |
| 03/068151 | August 2003 | WO |
| 2007/075915 | July 2007 | WO |
- Rudikoff et al. Single amino acid substitution altering antigen-binding specificity. Proc. Natl. Acad. Sci. USA, 79:1979-1983.
- Aldaz-Carroll, Lydia, et al., Epitope-Mapping Studies Define Two Major Neutralization Sites on the Vaccinia Virus Extracellular Enveloped Virus Glycoprotein B5R, Journal of Virology, 2005, 79:6260-6271.
- Bell, Edward, et al., Antibodies Against the Extracellular Enveloped Virus B5R Protein are Mainly Responsible for the EEV Neutralizing Capacity of Vaccinia Immune Globulin, Virology, 2004, 325:425-431.
- Davies, D. Hew, et al., Vaccinia Virus H3L Envelope Protein Is a Major Target of Neutralizing Antibodies in Humans and Elicits Protection Against Lethal Challenge in Mice, Journal of Virology, 2005, 79(18):11724-11733.
- Law, Mansun, et al., Antibody Neutralization of the Extracellular Enveloped Form of Vaccinia Virus, Virology, 2001, 280:132-142.
Type: Grant
Filed: Oct 6, 2008
Date of Patent: Jan 7, 2014
Patent Publication Number: 20100215658
Assignees: Kyowa Hakko Kirin Co., Ltd. (Tokyo), La Jolla Institute for Allergy and Immunology (La Jolla, CA)
Inventors: Shinichiro Kato (Chiba), Steve Granger (Encinitas, CA), Shane Crotty (San Diego, CA), Sandra Rickert (Encinitas, CA), Lilia Koriazova (San Diego, CA), Tomoyuki Tahara (San Diego, CA)
Primary Examiner: Jeffery Stucker
Assistant Examiner: Stuart W Snyder
Application Number: 12/682,539
International Classification: A61K 39/395 (20060101); A61K 39/42 (20060101);
